High efficiency base editors comprising gam

ABSTRACT

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins comprise a Gam protein, a napDNAbp, and a cytidine deaminase. In some embodiments, the fusion proteins further comprise a UGI domain. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a Gam protein, a cytidine deaminase and nucleic acid editing proteins or domains, are provided.

RELATED APPLICATIONS

This application is a divisional of and claims priority under 35 U.S.C.§ 120 to U.S. patent application U.S. Ser. No. 16/643,376, filed Feb.28, 2020, which is a national stage filing under 35 U.S.C. § 371 ofInternational PCT Application No. PCT/US2018/048969, filed Aug. 30,2018, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisionalapplication, U.S. Ser. No. 62/661,974, filed Apr. 24, 2018, and U.S.Provisional application, U.S. Ser. No. 62/551,938, filed Aug. 30, 2017,each of which is incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under HR0011-17-2-0049,EB022376 and GM118062 awarded by DARPA and National Institutes ofHealth. The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 18, 2022, is namedH082470260US03-SEQ-EPG and is 3,588,837 bytes in size.

BACKGROUND OF INVENTION

Traditional genome editing methods introduce a double-stranded DNA break(DSB) at a genomic target locus (14). The cellular response to a DSBlesion primarily proceeds through nonhomologous end joining (NHEJ) andrelated processes (15). Although NHEJ usually rejoins the two endsflanking the DSB, under typical genome editing conditions DSBs arecontinuously reintroduced, eventually resulting in the accumulation ofinsertions and deletions (indels) or translocations at the site of theDSB and disruption of the corresponding genomic locus (16). Activelydividing cells can also respond to DSBs by initiating homology-directedrepair (HDR) in the presence of a donor DNA template containing homologyto the regions surrounding the DSB, which allows researchers to moreprecisely and predictably manipulate genomes than is possible throughNHEJ (17). HDR-dependent genome editing is limited by low efficiencyarising from competition with NHEJ outcomes, and from the dependence ofHDR on mitosis (18).

The development of base editing, which enables the direct, irreversibleconversion of a C:G base pair to a T:A base pair in a programmablemanner without requiring HDR or the introduction of a DSB, was recentlyreported (1). Base editors contain a single-stranded DNA-specificcytidine deaminase enzyme tethered to a catalytically impaired Cas9protein and a base excision repair inhibitor (1, 4, 9, 10). The Cas9variant binds a genomic locus of interest, programmed by a correspondingguide RNA. Formation of the protein:RNA:DNA ternary “R-loop” complex(19) exposes a small (-5-nt) window of single-stranded DNA that servesas a substrate for the tethered cytidine deaminase enzyme. Cytidineswithin this window may be hydrolytically deaminated to uracils,resulting in G:U intermediates.

Base excision repair (BER) is the cell's primary response to G:Umismatches and is initiated by excision of the uracil by uracilN-glycosylase (UNG)(20). In an effort to protect the edited G:Uintermediate from excision by UNG, an 83-amino acid uracil glycosylaseinhibitor (UGI) was fused directly to the C-terminus of catalyticallydead Cas9 (dCas9) (1). To manipulate cellular DNA mismatch repairsystems into preferentially replacing the G in the G:U mismatch with anA, the Ala 840 amino acid in dCas9 was reverted to His, enabling theCas9 protein to nick the DNA strand opposite the newly formed uracil,resulting in much more efficient conversion of the G:U intermediate todesired A:U and A:T products (1). Combining these two engineeringefforts resulted in BE3, a single protein having a three-part fusion ofthe APOBEC1 cytidine deaminase enzyme tethered through a 16-amino acidlinker to S. pyogenes dCas9(A840H), which is covalently linked to UGIthrough a 4-amino acid linker(1). Subsequent to this report, thescientific community has used BE3 and related base editors for a widevariety of applications including plant genome editing, in vivomammalian genome editing, targeted mutagenesis, and knockout studies(2-13). The scope of base editing was expanded as described by reportingBE3 variants with altered PAM requirements (4), narrowed editing windows(4), reduced off-target editing (10), and small molecule dependence(21).

The programmable conversion of target C:G base pairs to T:A base pairswithout inducing double-stranded DNA breaks or requiringhomology-directed repair using engineered fusions of Cas9 variants andcytidine deaminases (1) was recently developed. Over the past year,third-generation base editors (e.g., BE3) and related technologies havebeen successfully used by many researchers in a wide range of organisms(2-13). At some loci, base editors such as BE3 give rise to undesiredbyproducts in which the target C:G base pair is converted into a G:C orA:T base pair, rather than the desired T:A product (2, 3, 6-8). Thus,there is a need to generate base editors that have improved performance,for example, base editors that have improved editing efficiency,improved product purity, and/or yield lower indel frequency.

BRIEF SUMMARY OF INVENTION

Provided herein are new base editors that convert C:G base pairs to T:Abase pairs with greater efficiency, higher product purity, and/orreduced indel frequencies than previously described base editors (e.g.,BE3). Some aspects of the disclosure are based on the discovery thatbase editors fused to a protein that binds to the ends of double strandbreaks, for example, the Gam protein of bacteriophage Mu, minimize theformation of undesired indels during base editing, and further increaseproduct purity. Thus, the disclosure provides new base editorscomprising proteins (e.g., Gam) that minimize the formation of indelsthat result from double strand breaks (DSB s).

Determinants of base editing product purity, which establish that UNGactivity is required for the formation of undesired byproducts, aredescribed herein. By analyzing individual DNA sequencing reads, it wasdiscovered that blocking UNG access to the uracil intermediate isimportant for target loci in which a single C is within the editingwindow in order to minimize undesired products. Using these insights, afourth-generation base editor, BE4 (e.g., SaBE4), was generated thatperforms base editing with higher efficiency and greatly improvedproduct purity compared to previously described base editors includingBE3. Further, additional base editors (e.g., BE3-Gam and BE4-Gam) weregenerated, which incorporate the dsDNA end-binding protein Gam tominimize the formation of undesired indels during base editing, and tofurther increase product purity.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show effects on base editing product purity of knocking outUNG. (FIG. 1A) Shows base editing results in HAP1 (UNG⁺) and HAP1 (UNG⁻)cells treated with BE3 as described in the Methods. The productdistribution among edited DNA sequencing reads (reads in which thetarget C is mutated) is shown. (FIG. 1B) Shows protospacer and PAM (PAMshown in bold) sequences of the genomic loci tested, with the target Csanalyzed in FIG. 1A are underlined. (FIG. 1C) shows frequency of inde1formation following treatment of HAP1 (UNG⁺) cells or HAP1 (UNG⁻) cellswith BE3. Values and error bars reflect the mean and s.d. of threeindependent biological replicates performed on different days.

FIGS. 2A-2E show fusion with Gam from bacteriophage Mu reduces indelfrequencies. (FIG. 2A) Shows schematic representations of thearchitectures of base editors, BE3-Gam and BE4-Gam. (FIG. 2B) Shows baseediting results of HEK293T cells that were treated with BE3, BE3-Gam,BE4, or BE4-Gam as described in the Methods. C-to-T base editingefficiencies are shown. (FIG. 2C) Shows frequency of indel formation(see Methods) following the treatment in FIG. 2B. (FIG. 2D) Shows theproduct distribution among edited DNA sequencing reads (reads in whichthe target C is mutated). (FIG. 2E) Shows recommended base editors whenprioritizing high editing efficiency, high product purity, and/or lowindel frequency. Values and error bars of BE3-Gam and BE4-Gam reflectthe mean and s.d. of three independent biological replicates performedon different days. Values and error bars of BE3 and BE4 reflect the meanand s.d. of six independent biological replicates performed on differentdays by two different researchers.

FIG. 3 shows BE4 induces lower indel frequencies than BE3, andTarget-AID exhibits similar product purities as CDA1-BE3. HEK293T cellswere treated with BE3, BE3-Gam, BE4, or BE4-Gam as described in theMethods. The ratio of editing efficiency to indel rate is calculated bydividing the percent of total sequencing reads in which the target C(indicated in red and with subscripts in FIG. 1B) is converted to T bythe frequency of indel formation (see Methods).

Definitions

As used herein and in the claims, the singular forms “a,” “an,” and“the” include the singular and the plural reference unless the contextclearly indicates otherwise. Thus, for example, a reference to “anagent” includes a single agent and a plurality of such agents.

The term “nucleic acid programmable DNA binding protein” or “napDNAbp”refers to a protein that associates with a nucleic acid (e.g., DNA orRNA), such as a guide nucleic acid (e.g., gRNA), that guides thenapDNAbp to a specific nucleic acid sequence, for example, byhybridinzing to the target nucleic acid sequence. For example, a Cas9protein can associate with a guide RNA that guides the Cas9 protein to aspecific DNA sequence is has complementary to the guide RNA. In someembodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. Insome embodiments, the napDNAbp is a Cas9 domain, for example, a nucleaseactive Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9(dCas9). Examples of nucleic acid programmable DNA binding proteinsinclude, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY,Cpf1, C2c1, C2c2, C2C3, and Argonaute. It should be appreciated,however, that nucleic acid programmable DNA binding proteins alsoinclude nucleic acid programmable proteins that bind RNA. For example,the napDNAbp may be associated with a nucleic acid that guides thenapDNAbp to an RNA. Other nucleic acid programmable DNA binding proteinsare also within the scope of this disclosure, though they may not bespecifically described in this disclosure.

In some embodiments, the napDNAby is an “RNA-programmable nuclease” or“RNA-guided nuclease.” The terms are used interchangeably herein andrefer to a nuclease that forms a complex with (e.g., binds or associateswith) one or more RNA(s) that is not a target for cleavage. In someembodiments, an RNA-programmable nuclease, when in a complex with anRNA, may be referred to as a nuclease:RNA complex. Typically, the boundRNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as acomplex of two or more RNAs, or as a single RNA molecule. gRNAs thatexist as a single RNA molecule may be referred to as single-guide RNAs(sgRNAs), though “gRNA” is also used to refer to guide RNAs that existas either single molecules or as a complex of two or more molecules.Typically, gRNAs that exist as a single RNA species comprise twodomains: (1) a domain that shares homology to a target nucleic acid(i.e., directs binding of a Cas9 complex to the target); and (2) adomain that binds a Cas9 protein. In some embodiments, domain (2)corresponds to a sequence known as a tracrRNA and comprises a stem-loopstructure. In some embodiments, domain (2) is identical or homologous toa tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), theentire contents of which is incorporated herein by reference. Otherexamples of gRNAs (e.g., those including domain 2) can be found in U.S.Provisional patent application, U.S. Ser. No. 61/874,682, filed Sep. 6,2013, entitled “Switchable Cas9 Nucleases And Uses Thereof,” and U.S.Provisional patent application, U.S. Ser. No. 61/874,746, filed Sep. 6,2013, entitled “Delivery System For Functional Nucleases,” the entirecontents of each are hereby incorporated by reference in their entirety.In some embodiments, a gRNA comprises two or more of domains (1) and(2), and may be referred to as an “extended gRNA.” For example, anextended gRNA will bind two or more Cas9 proteins and bind a targetnucleic acid at two or more distinct regions, as described herein. ThegRNA comprises a nucleotide sequence that complements a target site,which mediates binding of the nuclease/RNA complex to said target site,providing the sequence specificity of the nuclease:RNA complex. In someembodiments, the RNA-programmable nuclease is the (CRISPR-associatedsystem) Cas9 endonuclease, for example, Cas9 (Csn1) from Streptococcuspyogenes (see, e.g., “Complete genome sequence of an M1 strain ofStreptococcus pyogenes.” Ferretti J. J., McShan W. M., Ajdic D. J.,Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N.,Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., RenQ., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B.A.,McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNaseIII.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y.,Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonucleasein adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I.,Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), theentire contents of each of which are incorporated herein by reference.

Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNAhybridization to target DNA cleavage sites, these proteins are able totarget, in principle, any sequence specified by the guide RNA. Methodsof using RNA-programmable nucleases, such as Cas9, for site-specificcleavage (e.g., to modify a genome) are known in the art (see e.g.,Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems.Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genomeengineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al.,Efficient genome editing in zebrafish using a CRISPR-Cas system. Naturebiotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genomeediting in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al.,Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.Nucleic Acids Research (2013); Jiang, W. et al., RNA-guided editing ofbacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31,233-239 (2013); the entire contents of each of which are incorporatedherein by reference).

The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nucleasecomprising a Cas9 protein, or a fragment thereof (e.g., a proteincomprising an active, inactive, or partially active DNA cleavage domainof Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease isalso referred to sometimes as a casn1 nuclease or a CRISPR (clusteredregularly interspaced short palindromic repeat)-associated nuclease.CRISPR is an adaptive immune system that provides protection againstmobile genetic elements (viruses, transposable elements and conjugativeplasmids). CRISPR clusters contain spacers, sequences complementary toantecedent mobile elements, and target invading nucleic acids. CRISPRclusters are transcribed and processed into CRISPR RNA (crRNA). In typeII CRISPR systems correct processing of pre-crRNA requires atrans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) anda Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aidedprocessing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNAendonucleolytically cleaves linear or circular dsDNA targetcomplementary to the spacer. The target strand not complementary tocrRNA is first cut endonucleolytically, then trimmed 3′-5′exonucleolytically. In nature, DNA-binding and cleavage typicallyrequires protein and both RNAs. However, single guide RNAs (“sgRNA”, orsimply “gNRA”) can be engineered so as to incorporate aspects of boththe crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M.,Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science337:816-821(2012), the entire contents of which is hereby incorporatedby reference. Cas9 recognizes a short motif in the CRISPR repeatsequences (the PAM or protospacer adjacent motif) to help distinguishself versus non-self. Cas9 nuclease sequences and structures are wellknown to those of skill in the art (see, e.g., “Complete genome sequenceof an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J.,McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C.,Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., JiaH. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., CliftonS.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNAand host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M.,Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., CharpentierE., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K.,Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science337:816-821(2012), the entire contents of each of which are incorporatedherein by reference). Cas9 orthologs have been described in variousspecies, including, but not limited to, S. pyogenes and S. thermophilus.Additional suitable Cas9 nucleases and sequences will be apparent tothose of skill in the art based on this disclosure, and such Cas9nucleases and sequences include Cas9 sequences from the organisms andloci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA andCas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology10:5, 726-737; the entire contents of which are incorporated herein byreference. In some embodiments, a Cas9 nuclease has an inactive (e.g.,an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.

A nuclease-inactivated Cas9 protein may interchangeably be referred toas a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generatinga Cas9 protein (or a fragment thereof) having an inactive DNA cleavagedomain are known (See, e.g., Jinek et al., Science. 337:816-821(2012);Qi et al., “Repurposing CRISPR as an RNA-Guided Platform forSequence-Specific Control of Gene Expression” (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporatedherein by reference). For example, the DNA cleavage domain of Cas9 isknown to include two subdomains, the HNH nuclease subdomain and theRuvC1 subdomain. The HNH subdomain cleaves the strand complementary tothe gRNA, whereas the RuvC1 subdomain cleaves the non-complementarystrand. Mutations within these subdomains can silence the nucleaseactivity of Cas9. For example, the mutations D10A and H840A completelyinactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al.,Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)).In some embodiments, proteins comprising fragments of Cas9 are provided.For example, in some embodiments, a protein comprises one of two Cas9domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavagedomain of Cas9. In some embodiments, proteins comprising Cas9 orfragments thereof are referred to as “Cas9 variants.” A Cas9 variantshares homology to Cas9, or a fragment thereof. For example a Cas9variant is at least about 70% identical, at least about 80% identical,at least about 90% identical, at least about 95% identical, at leastabout 96% identical, at least about 97% identical, at least about 98%identical, at least about 99% identical, at least about 99.5% identical,or at least about 99.9% identical to wild type Cas9. In someembodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 or more amino acid changes compared to wild type Cas9. Insome embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., agRNA binding domain or a DNA-cleavage domain), such that the fragment isat least about 70% identical, at least about 80% identical, at leastabout 90% identical, at least about 95% identical, at least about 96%identical, at least about 97% identical, at least about 98% identical,at least about 99% identical, at least about 99.5% identical, or atleast about 99.9% identical to the corresponding fragment of wild typeCas9. In some embodiments, the fragment is at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% identical, at least 96%, at least 97%, at least98%, at least 99%, or at least 99.5% of the amino acid length of acorresponding wild type Cas9.

In some embodiments, the fragment is at least 100 amino acids in length.In some embodiments, the fragment is at least 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. Insome embodiments, wild type Cas9 corresponds to Cas9 from Streptococcuspyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO:1(nucleotide); SEQ ID NO:2 (amino acid)).

(SEQ ID NO: 1)ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGA GGTGACTGA(SEQ ID NO: 2)MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to, or comprises SEQ IDNO:3 (nucleotide) and/or SEQ ID NO: 4 (amino acid):

(SEQ ID NO: 3)ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA (SEQ ID NO: 4)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to Cas9 fromStreptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, SEQ ID NO:5 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 6(amino acid).

(SEQ ID NO: 5)ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGG AGGTGACTGA(SEQ ID NO: 6)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans(NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBIRefs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasmataiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref:NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); PsychroflexustorquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref:YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacterjejuni (NCBI Ref: YP_002344900.1) or Neisseria. meningitidis (NCBI Ref:YP_002342100.1) or to a Cas9 from any of the organisms listed in Example2.

In some embodiments, dCas9 corresponds to, or comprises in part or inwhole, a Cas9 amino acid sequence having one or more mutations thatinactivate the Cas9 nuclease activity. For example, in some embodiments,a dCas9 domain comprises D10A and/or H840A mutation.

dCas9 (D10A and H840A):

(SEQ ID NO: 7) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS

KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN

SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD  (single underline: HNH domain; double underline: RuvC domain).

In some embodiments, the Cas9 domain comprises a D10A mutation, whilethe residue at position 840 remains a histidine in the amino acidsequence provided in SEQ ID NO: 6, or at corresponding positions in anyof the amino acid sequences provided in SEQ ID NOs: 11-260. Withoutwishing to be bound by any particular theory, the presence of thecatalytic residue H840 restores the activity of the Cas9 to cleave thenon-edited (e.g., non-deaminated) strand containing a G opposite thetargeted C. Restoration of H840 (e.g., from A840) does not result in thecleavage of the target strand containing the C. Such Cas9 variants areable to generate a single-strand DNA break (nick) at a specific locationbased on the gRNA-defined target sequence, leading to repair of thenon-edited strand, ultimately resulting in a G to A change on thenon-edited strand. Briefly, the C of a C-G basepair can be deaminated toa U by a deaminase, e.g., an APOBEC deamonase. Nicking the non-editedstrand, having the G, facilitates removal of the G via mismatch repairmechanisms. UGI inhibits UDG, which prevents removal of the U.

In other embodiments, dCas9 variants having mutations other than D10Aand H840A are provided, which, e.g., result in nuclease inactivated Cas9(dCas9). Such mutations, by way of example, include other amino acidsubstitutions at D10 and H820, or other substitutions within thenuclease domains of Cas9 (e.g., substitutions in the HNH nucleasesubdomain and/or the RuvC1 subdomain). In some embodiments, variants orhomologues of dCas9 (e.g., variants of SEQ ID NO: 6) are provided whichare at least about 70% identical, at least about 80% identical, at leastabout 90% identical, at least about 95% identical, at least about 98%identical, at least about 99% identical, at least about 99.5% identical,or at least about 99.9% identical to SEQ ID NO: 6. In some embodiments,variants of dCas9 (e.g., variants of SEQ ID NO: 6) are provided havingamino acid sequences which are shorter, or longer than SEQ ID NO: 6, byabout 5 amino acids, by about 10 amino acids, by about 15 amino acids,by about 20 amino acids, by about 25 amino acids, by about 30 aminoacids, by about 40 amino acids, by about 50 amino acids, by about 75amino acids, by about 100 amino acids or more.

In some embodiments, Cas9 fusion proteins as provided herein comprisethe full-length amino acid sequence of a Cas9 protein, e.g., one of theCas9 sequences provided herein. In other embodiments, however, fusionproteins as provided herein do not comprise a full-length Cas9 sequence,but only a fragment thereof. For example, in some embodiments, a Cas9fusion protein provided herein comprises a Cas9 fragment, wherein thefragment binds crRNA and tracrRNA or sgRNA, but does not comprise afunctional nuclease domain, e.g., in that it comprises only a truncatedversion of a nuclease domain or no nuclease domain at all. Exemplaryamino acid sequences of suitable Cas9 domains and Cas9 fragments areprovided herein, and additional suitable sequences of Cas9 domains andfragments will be apparent to those of skill in the art.

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans(NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBIRefs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasmataiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref:NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); PsychroflexustorquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref:YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacterjejuni (NCBI Ref: YP_002344900.1); or Neisseria. meningitidis (NCBI Ref:YP_002342100.1).

The term “deaminase” or “deaminase domain,” as used herein, refers to aprotein or enzyme that catalyzes a deamination reaction. In someembodiments, the deaminase or deaminase domain is a cytidine deaminase,catalyzing the hydrolytic deamination of cytidine or deoxycytidine touridine or deoxyuridine, respectively. In some embodiments, thedeaminase or deaminase domain is a cytidine deaminase domain, catalyzingthe hydrolytic deamination of cytosine to uracil. In some embodiments,the deaminase or deaminase domain is a naturally-occurring deaminasefrom an organism, such as a human, chimpanzee, gorilla, monkey, cow,dog, rat, or mouse. In some embodiments, the deaminase or deaminasedomain is a variant of a naturally-occurring deaminase from an organismthat does not occur in nature. For example, in some embodiments, thedeaminase or deaminase domain is at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75% at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or at least 99.5% identical to a naturally-occurringdeaminase from an organism.

The term “Gam protein,” as used herein, refers generally to proteinscapable of binding to one or more ends of a double strand break of adouble stranded nucleic acid (e.g., double stranded DNA). In someembodiments, the Gam protein prevents or inhibits degradation of one ormore strands of a nucleic acid at the site of the double strand break.In some embodiments, a Gam protein is a naturally-occurring Gam proteinfrom bacteriophage Mu, or a non-naturally occurring variant thereof.

The term “effective amount,” as used herein, refers to an amount of abiologically active agent that is sufficient to elicit a desiredbiological response. For example, in some embodiments, an effectiveamount of a nuclease may refer to the amount of the nuclease that issufficient to induce cleavage of a target site specifically bound andcleaved by the nuclease. In some embodiments, an effective amount of afusion protein provided herein, e.g., of a fusion protein comprising anuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., adeaminase domain) may refer to the amount of the fusion protein that issufficient to induce editing of a target site specifically bound andedited by the fusion protein. As will be appreciated by the skilledartisan, the effective amount of an agent, e.g., a fusion protein, anuclease, a deaminase, a recombinase, a hybrid protein, a protein dimer,a complex of a protein (or protein dimer) and a polynucleotide, or apolynucleotide, may vary depending on various factors as, for example,on the desired biological response, e.g., on the specific allele,genome, or target site to be edited, on the cell or tissue beingtargeted, and on the agent being used.

The term “linker,” as used herein, refers to a chemical group or amolecule linking two molecules or moieties, e.g., two domains of afusion protein, such as, for example, a Cas9 domain (e.g., a Cas9nickase) and a nucleic acid editing domain (e.g., a deaminase domain).In some embodiments, a linker joins a gRNA binding domain of anRNA-programmable nuclease, including a Cas9 nuclease domain, and acatalytic domain of a nucleic-acid editing domain (e.g., a deaminasedomain). In some embodiments, a linker joins a Cas9 domain (e.g., a Cas9nickase) and a Gam protein. Typically, the linker is positioned between,or flanked by, two groups, molecules, or other moieties and connected toeach one via a covalent bond, thus connecting the two. In someembodiments, the linker is an amino acid or a plurality of amino acids(e.g., a peptide or protein). In some embodiments, the linker is anorganic molecule, group, polymer, or chemical moiety. In someembodiments, the linker is 5-100 amino acids in length, for example, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer orshorter linkers are also contemplated.

The term “mutation,” as used herein, refers to a substitution of aresidue within a sequence, e.g., a nucleic acid or amino acid sequence,with another residue, or a deletion or insertion of one or more residueswithin a sequence. Mutations are typically described herein byidentifying the original residue followed by the position of the residuewithin the sequence and by the identity of the newly substitutedresidue. Various methods for making the amino acid substitutions(mutations) provided herein are well known in the art, and are providedby, for example, Green and Sambrook, Molecular Cloning: A LaboratoryManual (4^(th) ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2012)).

The terms “nucleic acid” and “nucleic acid molecule,” as used herein,refer to a compound comprising a nucleobase and an acidic moiety, e.g.,a nucleoside, a nucleotide, or a polymer of nucleotides. Typically,polymeric nucleic acids, e.g., nucleic acid molecules comprising threeor more nucleotides are linear molecules, in which adjacent nucleotidesare linked to each other via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g. nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to an oligonucleotide chain comprising three or moreindividual nucleotide residues. As used herein, the terms“oligonucleotide” and “polynucleotide” can be used interchangeably torefer to a polymer of nucleotides (e.g., a string of at least threenucleotides). In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA. Nucleic acids may benaturally occurring, for example, in the context of a genome, atranscript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid,chromosome, chromatid, or other naturally occurring nucleic acidmolecule. On the other hand, a nucleic acid molecule may be anon-naturally occurring molecule, e.g., a recombinant DNA or RNA, anartificial chromosome, an engineered genome, or fragment thereof, or asynthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurringnucleotides or nucleosides. Furthermore, the terms “nucleic acid,”“DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g.,analogs having other than a phosphodiester backbone. Nucleic acids canbe purified from natural sources, produced using recombinant expressionsystems and optionally purified, chemically synthesized, etc. Whereappropriate, e.g., in the case of chemically synthesized molecules,nucleic acids can comprise nucleoside analogs such as analogs havingchemically modified bases or sugars, and backbone modifications. Anucleic acid sequence is presented in the 5′ to 3′ direction unlessotherwise indicated. In some embodiments, a nucleic acid is or comprisesnatural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine);chemically modified bases; biologically modified bases (e.g., methylatedbases); intercalated bases; modified sugars (e.g., 2′-fluororibose,ribose, 2′-deoxyribose, arabinose, and hexose); and/or modifiedphosphate groups (e.g., phosphorothioates and 5′-N-phosphoramiditelinkages).

The term “nucleic acid editing domain,” as used herein refers to aprotein or enzyme capable of making one or more modifications (e.g.,deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA).Exemplary nucleic acid editing domains include, but are not limited to adeaminase, a nuclease, a nickase, a recombinase, a methyltransferase, amethylase, an acetylase, an acetyltransferase, a transcriptionalactivator, or a transcriptional repressor domain. In some embodimentsthe nucleic acid editing domain is a deaminase (e.g., a cytidinedeaminase, such as an APOBEC or an AID deaminase).

The term “proliferative disease,” as used herein, refers to any diseasein which cell or tissue homeostasis is disturbed in that a cell or cellpopulation exhibits an abnormally elevated proliferation rate.Proliferative diseases include hyperproliferative diseases, such aspre-neoplastic hyperplastic conditions and neoplastic diseases.Neoplastic diseases are characterized by an abnormal proliferation ofcells and include both benign and malignant neoplasms. Malignantneoplasia is also referred to as cancer.

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof. Theterm “fusion protein” as used herein refers to a hybrid polypeptidewhich comprises protein domains from at least two different proteins.One protein may be located at the amino-terminal (N-terminal) portion ofthe fusion protein or at the carboxy-terminal (C-terminal) protein thusforming an “amino-terminal fusion protein” or a “carboxy-terminal fusionprotein,” respectively. A protein may comprise different domains, forexample, a nucleic acid binding domain (e.g., the gRNA binding domain ofCas9 that directs the binding of the protein to a target site) and anucleic acid cleavage domain or a catalytic domain of a nucleic-acidediting protein. In some embodiments, a protein comprises aproteinaceous part, e.g., an amino acid sequence constituting a nucleicacid binding domain, and an organic compound, e.g., a compound that canact as a nucleic acid cleavage agent. In some embodiments, a protein isin a complex with, or is in association with, a nucleic acid, e.g., RNA.Any of the proteins provided herein may be produced by any method knownin the art. For example, the proteins provided herein may be producedvia recombinant protein expression and purification, which is especiallysuited for fusion proteins comprising a peptide linker. Methods forrecombinant protein expression and purification are well known, andinclude those described by Green and Sambrook, Molecular Cloning: ALaboratory Manual (4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2012)), the entire contents of which areincorporated herein by reference.

The term “subject,” as used herein, refers to an individual organism,for example, an individual mammal. In some embodiments, the subject is ahuman. In some embodiments, the subject is a non-human mammal. In someembodiments, the subject is a non-human primate. In some embodiments,the subject is a rodent. In some embodiments, the subject is a sheep, agoat, a cattle, a cat, or a dog. In some embodiments, the subject is avertebrate, an amphibian, a reptile, a fish, an insect, a fly, or anematode. In some embodiments, the subject is a research animal. In someembodiments, the subject is genetically engineered, e.g., a geneticallyengineered non-human subject. The subject may be of either sex and atany stage of development.

The term “target site” refers to a sequence within a nucleic acidmolecule that is deaminated by a deaminase or a fusion proteincomprising a deaminase, (e.g., a Gam-nCas9-deaminase fusion proteinprovided herein).

The terms “treatment,” “treat,” and “treating” refer to a clinicalintervention aimed to reverse, alleviate, delay the onset of, or inhibitthe progress of a disease or disorder, or one or more symptoms thereof,as described herein. As used herein, the terms “treatment,” “treat,” and“treating” refer to a clinical intervention aimed to reverse, alleviate,delay the onset of, or inhibit the progress of a disease or disorder, orone or more symptoms thereof, as described herein. In some embodiments,treatment may be administered after one or more symptoms have developedand/or after a disease has been diagnosed. In other embodiments,treatment may be administered in the absence of symptoms, e.g., toprevent or delay onset of a symptom or inhibit onset or progression of adisease. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors).Treatment may also be continued after symptoms have resolved, forexample, to prevent or delay their recurrence.

The term “recombinant” as used herein in the context of proteins ornucleic acids refers to proteins or nucleic acids that do not occur innature, but are the product of human engineering. For example, in someembodiments, a recombinant protein or nucleic acid molecule comprises anamino acid or nucleotide sequence that comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, or atleast seven mutations as compared to any naturally occurring sequence.

The term “pharmaceutical composition,” as used herein, refers to acomposition that can be administrated to a subject in the context oftreatment of a disease or disorder. In some embodiments, apharmaceutical composition comprises an active ingredient, e.g., anuclease or a nucleic acid encoding a nuclease, and a pharmaceuticallyacceptable excipient.

The term “base editor (BE),” or “nucleobase editor (NBE),” as usedherein, refers to an agent comprising a polypeptide that is capable ofmaking a modification to a base (e.g., A, T, C, G, or U) within anucleic acid sequence (e.g., DNA or RNA). In some embodiments, the baseeditor is capable of deaminating a base within a nucleic acid. In someembodiments, the base editor is capable of deaminating a base within aDNA molecule. In some embodiments, the base editor is capable ofdeaminating a cytosine (C) in DNA. In some embodiments, the base editoris a fusion protein comprising a Gam protein, a nucleic acidprogrammable DNA binding protein (napDNAbp), and a cytidine deaminasedomain. In some embodiments, the base editor comprises a Cas9 (e.g.,dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonauteprotein fused to a cytidine deaminase. In some embodiments, the baseeditor comprises a Cas9 nickase (nCas9) fused to a cytidine deaminase.In some embodiments, the base editor comprises a nuclease-inactive Cas9(dCas9) fused to a cytidine deaminase. In some embodiments, the baseeditor is fused to a protein that binds to one or more ends of a doublestrand break in a double stranded nucleic acid (e.g., DNA or RNA). Insome embodiments, the base editor is fused to an inhibitor of baseexcision repair, for example, a UGI domain. In some embodiments, thebase editor comprises a Gam protein, fused to a CasX protein, which isfused to a cytidine deaminase. In some embodiments, the base editorcomprises a Gam protein, fused to a CasY protein, which is fused to acytidine deaminase. In some embodiments, the base editor comprises a Gamprotein, fused to a Cpf1 protein, which is fused to a cytidinedeaminase. In some embodiments, the base editor comprises a Gam protein,fused to a C2c1 protein, which is fused to a cytidine deaminase. In someembodiments, the base editor comprises a Gam protein, fused to a C2c2protein, which is fused to a cytidine deaminase. In some embodiments,the base editor comprises a Gam protein, fused to a C2c3 protein, whichis fused to a cytidine deaminase. In some embodiments, the base editorcomprises a Gam protein, fused to an Argonaute protein, which is fusedto a cytidine deaminase.

The term “uracil glycosylase inhibitor” or “UGI,” as used herein, refersto a protein that is capable of inhibiting a uracil-DNA glycosylasebase-excision repair enzyme.

The term “Cas9 nickase,” as used herein, refers to a Cas9 protein thatis capable of cleaving only one strand of a duplexed nucleic acidmolecule (e.g., a duplexed DNA molecule). In some embodiments, a Cas9nickase comprises a D10A mutation and has a histidine at position H840of SEQ ID NO: 6, or a corresponding mutation in any of SEQ ID NOs:11-260. For example, a Cas9 nickase may comprise the amino acid sequenceas set forth in SEQ ID NO: 8. Such a Cas9 nickase has an active HNHnuclease domain and is able to cleave the non-targeted strand of DNA,i.e., the strand bound by the gRNA. Further, such a Cas9 nickase has aninactive RuvC nuclease domain and is not able to cleave the targetedstrand of the DNA, i.e., the strand where base editing is desired.

Exemplary Cas9 nickase (Cloning vector pPlatTET-gRNA2; Accession No.BAV54124).

(SEQ ID NO: 8) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

DETAILED DESCRIPTION OF INVENTION

Some aspects of this disclosure provide fusion proteins that comprise adomain capable of binding to one or more ends of a double strand breakof a nucleic acid (e.g., a Gam protein); a domain capable of binding toa nucleotide sequence (e.g., a Cas9, or a Cpf1 protein) and an enzymedomain, for example, a DNA-editing domain, such as, e.g., a deaminasedomain. The deamination of a nucleobase by a deaminase can lead to apoint mutation at the respective residue, which constitutes nucleic acidediting. Fusion proteins comprising a Gam protein, or variant thereof, anapDNAbp domain, or variant thereof, and a DNA editing domain, orvariant thereof, can thus be used for the targeted editing of nucleicacid sequences. Such fusion proteins are useful for targeted editing ofDNA in vitro, e.g., for the generation of mutant cells or animals; forthe introduction of targeted mutations, e.g., for the correction ofgenetic defects in cells ex vivo, e.g., in cells obtained from a subjectthat are subsequently re-introduced into the same or another subject;and for the introduction of targeted mutations, e.g., the correction ofgenetic defects or the introduction of deactivating mutations indisease-associated genes in a subject. Such fusion proteins are usefulfor targeted editing of DNA that generate fewer indels (e.g., <0.01%,0.05%, 0.1%, 0.2%, 0.3%, 0.5%, or 1% indels) than other base editors(e.g., BE3 or BE4). Typically, the Cas9 domain of the fusion proteinsdescribed herein does not have full nuclease activity but instead is aCas9 nickase, a Cas9 fragment, or a nuclease-inactive Cas9 domain(dCas9). Methods for the use of Cas9 fusion proteins as described hereinare also provided.

Some aspects of the disclosure are based on the discovery that fusingBE3 or BE4 to Gam, a bacteriophage Mu protein that binds double-strandedDNA breaks, greatly reduces indel formation during base editing, in mostcases to below 0.5%. BE4 and BE4-Gam represent the state-of-the-art inC:G to T:A base editing.

Fusion Proteins Comprising Gam

Some aspects of the disclosure provide fusion proteins comprising a Gamprotein. Some aspects of the disclosure provide base editors thatfurther comprise a Gam protein. Base editors are known in the art andhave been described previously, for example, in U.S. Patent ApplicationPublication Nos.: US-2015-0166980, published Jun. 18, 2015;US-2015-0166981, published Jun. 18, 2015; US-2015-0166984, publishedJun. 18, 2015; US-2015-01669851, published Jun. 18, 2015;US-2016-0304846, published Oct. 20, 2016; US-2017-0121693-A1, publishedMay 4, 2017; and PCT Application publication Nos.: WO2015089406,published Jun. 18, 2015; and WO2017070632, published Apr. 27, 2017; theentire contents of each of which are hereby incorporated by reference. Askilled artisan would understand, based on the disclosure, how to makeand use base editors that further comprise a Gam protein.

In some embodiments, the disclosure provides fusion proteins comprisinga nucleic acid programmable DNA binding protein (napDNAbp) and a Gamprotein. In some embodiments, the disclosure provides fusion proteinscomprising a cytidine deaminase domain and a Gam protein. In someembodiments, the disclosure provides fusion proteins comprising a UGIdomain and a Gam protein. In some embodiments, the disclosure providesfusion proteins comprising a nucleic acid programmable DNA bindingprotein (napDNAbp), a cytidine deaminase domain and a Gam protein. Insome embodiments, the disclosure provides fusion proteins comprising anucleic acid programmable DNA binding protein (napDNAbp), a cytidinedeaminase domain a Gam protein and a UGI domain.

In some embodiments, the Gam protein is a protein that binds to doublestrand breaks in DNA and prevents or inhibits degradation of the DNA atthe double strand breaks. In some embodiments, the Gam protein isencoded by the bacteriophage Mu, which binds to double stranded breaksin DNA. Without wishing to be bound by any particular theory, Mutransposes itself between bacterial genomes and uses Gam to protectdouble stranded breaks in the transposition process. Gam can be used toblock homologous recombination with sister chromosomes to repair doublestrand breaks, sometimes leading to cell death. The survival of cellsexposed to UV is similar for cells expression Gam and cells where therecB is mutated. This indicates that Gam blocks DNA repair (Cox, 2013).The Gam protein can thus promote Cas9-mediated killing (Cui et al.,2016). GamGFP is used to label double stranded breaks, although this canbe difficult in eukaryotic cells as the Gam protein competes withsimilar eukaryotic protein Ku (Shee et al., 2013).

Gam is related to Ku70 and Ku80, two eukaryotic proteins involved innon-homologous DNA end-joining (Cui et al., 2016). Gam has sequencehomology with both subunits of Ku (Ku70 and Ku80), and can have asimilar structure to the core DNA-binding region of Ku. Orthologs to MuGam are present in the bacterial genomes of Haemophilus influenzae,Salmonella typhi, Neisseria meningitidis and the enterohemorrhagicO157:H7 strain of E. coli (d′Adda di Fagagna et al., 2003). Gam proteinshave been described previously, for example, in COX, Proteins pinpointdouble strand breaks. eLife. 2013; 2: e01561; CUI et al., Consequencesof Cas9 cleavage in the chromosome of Escherichia coli. Nucleic AcidsRes. 2016 May 19; 44(9):4243-51. doi: 10.1093/nar/gkw223. Epub 2016 Apr.8; D′ADDA DI FAGAGNA et al., The Gam protein of bacteriophage Mu is anorthologue of eukaryotic Ku. EMBO Rep. 2003 January;4(1):47-52; and SHEEet al., Engineered proteins detect spontaneous DNA breakage in human andbacterial cells. Elife. 2013 Oct. 29; 2:e01222. doi: 10.7554/eLife.01222; the contents of each of which are incorporated herein byreference.

In some embodiments, the Gam protein is a protein that binds doublestrand breaks in DNA and prevents or inhibits degradation of the DNA atthe double strand breaks. In some embodiments, the Gam protein is anaturally occurring Gam protein from any organism (e.g., a bacterium),for example, any of the organisms provided herein. In some embodiments,the Gam protein is a variant of a naturally-occurring Gam protein froman organism. In some embodiments, the Gam protein does not occur innature. In some embodiments, the Gam protein is at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75% at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or at least 99.5% identical to anaturally-occurring Gam protein. In some embodiments, the Gam protein isat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75% at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to any of the Gam proteins provided herein (e.g., SEQ ID NO:9). Exemplary Gam proteins are provided below. In some embodiments, theGam protein comprises any of the Gam proteins provided herein (e.g., SEQID NO: 9). In some embodiments, the Gam protein is a truncated versionof any of the Gam proteins provided herein. In some embodiments, thetruncated Gam protein is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relativeto a full-length Gam protein. In some embodiments, the truncated Gamprotein may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to afull-length Gam protein. In some embodiments, the Gam protein does notcomprise an N-terminal methionine.

In some embodiments, the Gam protein comprises an amino acid sequencethat is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95, 98%, 99%, or99.5% identical to any of the Gam proteins provided herein. In someembodiments, the Gam protein comprises an amino acid sequence that has1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutationscompared to any one of the Gam Proteins provided herein. In someembodiments, the Gam protein comprises an amino acid sequence that hasat least 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 110, at least120, at least 130, at least 140, at least 150, at least 160, or at least170, identical contiguous amino acid residues as compared to any of theGam proteins provided herein. In some embodiments, the Gam proteincomprises the amino acid sequence of any of the Gam proteins providedherein. In some embodiments, the Gam protein comprises the amino acidsequence of SEQ ID NO: 9. In some embodiments, the Gam protein consistsof the any of the Gam proteins provided herein (e.g., SEQ ID NO: 9). Insome embodiments, the Gam protein comprises the amino acid sequence ofSEQ ID NO: 10 (i.e., contains an N-terminal methionine residue). In someembodiments, the Gam protein comprises the amino acid sequence of anyone of SEQ ID NOs: 261-283.

Gam form bacteriophage Mu (SEQ ID NO: 9)AKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIPFEQEAGI >WP_001107930.1 MULTISPECIES: host-nuclease inhibitor protein Gam[Enterobacteriaceae] (SEQ ID NO: 10)MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIP FEQEAGI>CAA27978.1 unnamed protein product [Escherichia virus Mu](SEQ ID NO: 261) MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFVRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSII PFEQEAGI>WP_001107932.1 host-nuclease inhibitor protein Gam [Escherichia coli](SEQ ID NO: 262) MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPLKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSII PFEQEAGI>WP_061335739.1 host-nuclease inhibitor protein Gam [Escherichia coli](SEQ ID NO: 263) MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLITGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIPFEQEAGI >WP_001107937.1 MULTISPECIES: host-nuclease inhibitor protein Gam[Enterobacteriaceae] >EJL11163.1 bacteriophage Mu Gam like family protein [Shigellasonnei str. Moseley] >CSO81529.1 host-nuclease inhibitor protein [Shigella sonnei]>OCE38605.1 host-nuclease inhibitor protein Gam [Shigella sonnei] >SJK50067.1 host-nuclease inhibitor protein [Shigella sonnei] >SJK19110.1 host-nuclease inhibitor protein[Shigella sonnei] >SIY81859.1 host-nuclease inhibitor protein [Shigella sonnei]>SJJ34359.1 host-nuclease inhibitor protein [Shigella sonnei] >SJK07688.1 host-nucleaseinhibitor protein [Shigella sonnei] >SJI95156.1 host-nuclease inhibitor protein [Shigellasonnei] >SIY86865.1 host-nuclease inhibitor protein [Shigella sonnei] >SJJ67303.1 host-nuclease inhibitor protein [Shigella sonnei] >SJJ18596.1 host-nuclease inhibitor protein[Shigella sonnei] >SIX52979.1 host-nuclease inhibitor protein [Shigella sonnei]>SJD05143.1 host-nuclease inhibitor protein [Shigella sonnei] >SJD37118.1 host-nucleaseinhibitor protein [Shigella sonnei] >SJE51616.1 host-nuclease inhibitor protein [Shigellasonnei] (SEQ ID NO: 264)MAKPAKRIRNAAAAYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGlEDFSIIP FEQEAGI>WP_089552732.1 host-nuclease inhibitor protein Gam [Escherichia coli](SEQ ID NO: 265) MAKPAKRIKNAAAAYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETISKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGlEDFSIIP FEQEAGI>WP_042856719.1 host-nuclease inhibitor protein Gam [Escherichia coli]>CDL02915.1 putative host-nuclease inhibitor protein [Escherichia coli IS35](SEQ ID NO: 266) MAKPAKRIKNAAAAYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIADITEKYASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSII PFEQEAGI>WP_001129704.1 host-nuclease inhibitor protein Gam [Escherichia coli]>EDU62392.1 bacteriophage Mu Gam like protein [Escherichia coli 53638](SEQ ID NO: 267) MAKSAKRIRNAAAAYVPQSRDAVVCDIRRIGNLQREAARLETEMNDAIAEITEKFAARIAPLKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINREAILLEPKAVAGVAGITVKSGIEDFSIIPFEQDAGI >WP_001107936.1 MULTISPECIES: host-nuclease inhibitor protein Gam[Enterobacteriaceae] >EGI94970.1 host-nuclease inhibitor protein gam [Shigella boydii5216-82] >C5R34065.1 host-nuclease inhibitor protein [Shigella sonnei] >CSQ65903.1host-nuclease inhibitor protein [Shigella sonnei] >CSQ94361.1 host-nuclease inhibitorprotein [Shigella sonnei] >SJK23465.1 host-nuclease inhibitor protein [Shigella sonnei]>SJB59111.1 host-nuclease inhibitor protein [Shigella sonnei] >SJI55768.1 host-nucleaseinhibitor protein [Shigella sonnei] >SJI56601.1 host-nuclease inhibitor protein [Shigellasonnei] >SJJ20109.1 host-nuclease inhibitor protein [Shigella sonnei] >SJJ54643.1 host-nuclease inhibitor protein [Shigella sonnei] >SJI29650.1 host-nuclease inhibitor protein[Shigella sonnei] >SIZ53226.1 host-nuclease inhibitor protein [Shigella sonnei]>SJA65714.1 host-nuclease inhibitor protein [Shigella sonnei] >SJJ21793.1 host-nucleaseinhibitor protein [Shigella sonnei] >SJD61405.1 host-nuclease inhibitor protein [Shigellasonnei] >SJJ14326.1 host-nuclease inhibitor protein [Shigella sonnei] >SIZ57861.1 host-nuclease inhibitor protein [Shigella sonnei] >SJD58744.1 host-nuclease inhibitor protein[Shigella sonnei] >SJD84738.1 host-nuclease inhibitor protein [Shigella sonnei]>SJJ51125.1 host-nuclease inhibitor protein [Shigella sonnei] >SJD01353.1 host-nucleaseinhibitor protein [Shigella sonnei] >SJE63176.1 host-nuclease inhibitor protein [Shigellasonnei] (SEQ ID NO: 268)MAKPAKRIRNAAAAYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGlEDFSIIP FEQDAGI>WP_050939550.1 host-nuclease inhibitor protein Gam [Escherichia coli]>KNF77791.1 host-nuclease inhibitor protein Gam [Escherichia coli](SEQ ID NO: 269) MAKPAKRIKNAAAAYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRLRPPSVSIRGVDAVMETLERLGLQRFICTKQEINKEAILLEPKVVAGVAGITVKSGlEDFSIIP FEQEAGI>WP_085334715.1 host-nuclease inhibitor protein Gam [Escherichia coli]>OSC16757.1 host-nuclease inhibitor protein Gam [Escherichia coli](SEQ ID NO: 270) MAKPVKRIRNAAAAYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGIQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGlEDFSIIP FEQEAGI>WP_065226797.1 host-nuclease inhibitor protein Gam [Escherichia coli]>AN088858.1 host-nuclease inhibitor protein Gam [Escherichia coli] >ANO89006.1 host-nuclease inhibitor protein Gam [Escherichia coli] (SEQ ID NO: 271)MAKPAKRIRNAAAAYVPQSRDAVVCDIRWIGDLQREAVRLETEMNDAIAEITEKYASRIAPLKTRIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSI IPFEQEAGI>WP_032239699.1 host-nuclease inhibitor protein Gam [Escherichia coli]>KDU26235.1 bacteriophage Mu Gam like family protein [Escherichia coli 3-373-03_S4_C2] >KDU49057.1 bacteriophage Mu Gam like family protein [Escherichia coli 3-373-03_S4_C1] >KEL21581.1 bacteriophage Mu Gam like family protein [Escherichia coli3-373-03_S4_C3] (SEQ ID NO: 272)MAKSAKRIRNAAATYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGIQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSG1EDFSIIP FEQEAGI>WP_080172138.1 host-nuclease inhibitor protein Gam [Salmonella enterica](SEQ ID NO: 273) MAKSAKRIKSAAATYVPQSRDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKSANLVTGDVQWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSII PFEQEAGI>WP_077134654.1 host-nuclease inhibitor protein Gam [Shigella sonnei]>SIZ51898.1 host-nuclease inhibitor protein [Shigella sonnei] >SJK07212.1 host-nucleaseinhibitor protein [Shigella sonnei] (SEQ ID NO: 274)MAKSAKRIRNAAAAYVPQSRDAVVCDIRRIGNLQREAARLETEMNDAIAEITEKYASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSG1EDFSIIP FEQDAGI>WP_000261565.1 host-nuclease inhibitor protein Gam [Shigella flexneri]>EGK20651.1 host-nuclease inhibitor protein gam [Shigella flexneri K-272] >EGK34753.1host-nuclease inhibitor protein gam [Shigella flexneri K-227](SEQ ID NO: 275) MVVSAIASTPHDAVVCDIRRIGDLQREAARLETEMNDAIAEITEKDASQIAPLKTSIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRQRPPSVSIRGVDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIPFEQEAGI>ASG63807.1 host-nuclease inhibitor protein Gam [Kluyvera georgiana](SEQ ID NO: 276) MVSKPKRIKAAAANYVSQSRDAVITDIRKIGDLQREATRLESAMNDEIAVITEKYAGLIKPLKADVEMLSKGVQGWCEANRDDLTSNGKVKTANLVTGDIQWRIRPPSVSVRGPDAVMETLTRLGLSRFIRTKQEINKEAILNEPLAVAGVAGITVKSGIEDFSIIP FEQTADI>WP_078000363.1 host-nuclease inhibitor protein Gam [Edwardsiella tarda](SEQ ID NO: 277) MASKPKRIKSAAANYVSQSRDAVIIDIRKIGDLQREATRLESAMNDEIAVITEKYAGLIKPLKADVEMLSKGVQGWCEANRDELTCNGKVKTANLVTGDIQWRIRPPSVSVRGPDSVMETLLRLGLSRFIRTKQEINKEAILNEPLAVAGVAGITVKTGVEDFSIIP FEQTADI>WP_047389411.1 host-nuclease inhibitor protein Gam [Citrobacter freundii]>KGY86764.1 host-nuclease inhibitor protein Gam [Citrobacter freundii] >OIZ37450.1host-nuclease inhibitor protein Gam [Citrobacter freundii](SEQ ID NO: 278) MVSKPKRIKAAAANYVSQSKEAVIADIRKIGDLQREATRLESAMNDEIAVITEKYAGLIKPLKTDVEILSKGVQGWCEANRDELTSNGKVKTANLVTGDIQWRIRPPSVAVRGPDAVMETLLRLGLSRFIRTKQEINKEAILNEPLAVAGVAGITVKSGVEDFSII PFEQTADI>WP_058215121.1 host-nuclease inhibitor protein Gam [Salmonella enterica]>KSU39322.1 host-nuclease inhibitor protein Gam [Salmonella enterica subsp. enterica]>OHJ24376.1 host-nuclease inhibitor protein Gam [Salmonella enterica] >ASG15950.1host-nuclease inhibitor protein Gam [Salmonella enterica subsp. enterica serovarMacclesfield str. S-1643] (SEQ ID NO: 279)MASKPKRIKAAAALYVSQSREDVVRDIRMIGDFQREIVRLETEMNDQIAAVTLKYADKIKPLQEQLKTLSEGVQNWCEANRSDLTNGGKVKTANLVTGDVQWRVRPPSVTVRGVDSVMETLRRLGLSRFIRIKEEINKEAILNEPGAVAGVAGITVKSGVEDFS IIPFEQSATN>WP_016533308.1 phage host-nuclease inhibitor protein Gam [Pasteurellamultocida] >EPE65165.1 phage host-nuclease inhibitor protein Gam [Pasteurella multocidaP1933] >ESQ71800.1 host-nuclease inhibitor protein Gam [Pasteurella multocida subsp.multocida P1062] >ODS44103.1 host-nuclease inhibitor protein Gam [Pasteurellamultocida] >OPC87246.1 host-nuclease inhibitor protein Gam [Pasteurella multocidasubsp. multocida] >OPC98402.1 host-nuclease inhibitor protein Gam [Pasteurellamultocida subsp. multocida] (SEQ ID NO: 280)MAKKATRIKTTAQVYVPQSREDVASDIKTIGDLNREITRLETEMNDKIAEITESYKGQFSPIQERIKNLSTGVQFWAEANRDQITNGGKTKTANLITGEVSWRVRNPSVKITGVDSVLQNLKIHGLTKFIRVKEEINKEAILNEKHEVAGIAGIKVVSGVEDFVITPF EQEI>WP_005577487.1 host-nuclease inhibitor protein Gam [Aggregatibacteractinomycetemcomitans] >EHK90561.1 phage host-nuclease inhibitor protein Gam[Aggregatibacter actinomycetemcomitans RhAA1] >KNE77613.1 host-nuclease inhibitorprotein Gam [Aggregatibacter actinomycetemcomitans RhAA1](SEQ ID NO: 281) MAKSATRVKATAQIYVPQTREDAAGDIKTIGDLNREVARLEAEMNDKIAAITEDYKDKFAPLQERIKTLSNGVQYWSEANRDQITNGGKTKTANLVTGEVSWRVRNPSVKVTGVDSVLQNLRIHGLERFIRTKEEINKEAILNEKSAVAGIAGIKVITGVEDFVIT PFEQEAA>WP_090412521.1 host-nuclease inhibitor protein Gam [Nitrosomonas halophila]>SDX89267.1 Mu-like prophage host-nuclease inhibitor protein Gam [Nitrosomonashalophila] (SEQ ID NO: 282)MARNAARLKTKSIAYVPQSRDDAAADIRKIGDLQRQLTRTSTEMNDAIAAITQNFQPRMDAIKEQINLLQAGVQGYCEAHRHALTDNGRVKTANLITGEVQWRQRPPSVSIRGQQVVLETLRRLGLERFIRTKEEVNKEAILNEPDEVRGVAGLNVITGVEDFVI TPFEQEQP >WP_077926574.1 host-nuclease inhibitor protein Gam [Wohlfahrtiimonas larvae](SEQ ID NO: 283) MAKKRIKAAATVYVPQSKEEVQNDIREIGDISRKNERLETEMNDRIAEITNEYAPKFEVNKVRLELLTKGVQSWCEANRDDLTNS GKVKSANLVTGKVEWRQRPPSISVKGMDAVIEWLQDSKYQRFLRTKVEVNKEAMLNEPEDAKTIPGITIKSGIEDFAITP FEQEAGV 

Nucleic Acid Programmable DNA Binding Proteins

Some aspects of the disclosure provide nucleic acid programmable DNAbinding proteins, which may be used to guide a protein, such as a baseeditor, to a specific nucleic acid (e.g., DNA or RNA) sequence. Itshould be appreciated that any of the fusion proteins (e.g., baseeditors) provided herein may include any nucleic acid programmable DNAbinding protein (napDNAbp). For example, any of the fusion proteinsdescribed herein that include a Cas9 domain, can use another napDNAbp,such as CasX, CasY, Cpf1, C2c1, C2c2, C2c3, and Argonaute, in place ofthe Cas9 domain. Nucleic acid programmable DNA binding proteins include,without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1,C2c1, C2c2, C2C3, and Argonaute. One example of a nucleic acidprogrammable DNA-binding protein that has a different PAM specificitythan Cas9 is Clustered Regularly Interspaced Short Palindromic Repeatsfrom Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is alsoa class 2 CRISPR effector. It has been shown that Cpf1mediates robustDNA interference with features distinct from Cas9. Cpf1 is a singleRNA-guided endonuclease lacking tracrRNA, and it utilizes a T-richprotospacer-adjacent motif (TTN, TTTN (SEQ ID NO: 284), or YTN).Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break.Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus andLachnospiraceae are shown to have efficient genome-editing activity inhuman cells. Cpf1 proteins are known in the art and have been describedpreviously, for example, Yamano et al., “Crystal structure of Cpf1 incomplex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; theentire contents of which are incorporated herein by reference.

Also useful in the present compositions and methods arenuclease-inactive Cpf1 (dCpf1) variants that may be used as a guidenucleotide sequence-programmable DNA-binding protein domain. The Cpf1protein has a RuvC-like endonuclease domain that is similar to the RuvCdomain of Cas9 but does not have a HNH endonuclease domain, and theN-terminal of Cpf1 does not have the alpha-helical recognition lobe ofCas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which isincorporated herein by reference) that, the RuvC-like domain of Cpf1 isresponsible for cleaving both DNA strands and inactivation of theRuvC-like domain inactivates Cpf1 nuclease activity. For example,mutations corresponding to D917A, E1006A, or D1255A in Francisellanovicida Cpf1 (SEQ ID NO: 753) inactivate Cpf1 nuclease activity. Insome embodiments, the dead Cpf1 (dCpf1) comprises mutationscorresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A,E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 285. It is to beunderstood that any mutations, e.g., substitution mutations, deletions,or insertions, that inactivate the RuvC domain of Cpf1, may be used inaccordance with the present disclosure.

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) of any of the fusion proteins provided herein is a Cpf1protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase(nCpf1). In some embodiments, the Cpf1 protein is a nuclease inactiveCpf1 (dCpf1). In some embodiments, the Cpf1, the nCpf1, or the dCpf1comprises an amino acid sequence that is at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to any one of SEQ ID NOs: 285-292 or 293-303. In someembodiments, the dCpf1 comprises an amino acid sequence that is at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or at least 99.5% identical to any one of SEQ ID NOs: 285-292, andcomprises mutations corresponding to D917A, E1006A, D1255A,D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQID NO: 376. In some embodiments, the dCpf1 protein comprises an aminoacid sequence of any one SEQ ID NOs: 285-292. It should be appreciatedthat Cpf1 from other species may also be used in accordance with thepresent disclosure.

Wild type Francisella novicida Cpf1 (SEQ ID NO: 285) (D917, E1006,and D1255 are bolded and underlined) (SEQ ID NO: 285)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 D917A (SEQ ID NO: 286) (A917, E1006, andD1255 are bolded and underlined) (SEQ ID NO: 286)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 E1006A (SEQ ID NO: 287) (D917, A1006, andD1255 are bolded and underlined) (SEQ ID NO: 287)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 D1255A (SEQ ID NO: 288) (D917, E1006, andA1255 are bolded and underlined) (SEQ ID NO: 288)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 D917A/E1006A (SEQ ID NO: 289) (A917,A1006, and D1255 are bolded and underlined) (SEQ ID NO: 289)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 D917A/D1255A (SEQ ID NO: 290) (A917,E1006, and A1255 are bolded and underlined) (SEQ ID NO: 290)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 E1006A/D1255A (SEQ ID NO: 291) (D917,A1006, and A1255 are bolded and underlined) (SEQ ID NO: 291)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNNFrancisella novicida Cpf1 D917A/E1006A/D1255A (SEQ ID NO: 292)(A917, A1006, and A1255 are bolded and underlined) (SEQ ID NO: 292)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQN RNN

In some embodiments, the nucleic acid programmable DNA binding proteinis a Cpf1 protein from an Acidaminococcus species (AsCpf1). Cpf1proteins form Acidaminococcus species have been described previously andwould be apparent to the skilled artisan. Exemplary Acidaminococcus Cpf1proteins (AsCpf1) include, without limitation, any of the AsCpf1proteins provided herin

Wild-type AsCpf1- Residue R912 is indicated in bold underlining andresidues 661-667 are indicated in italics and underlining.(SEQ ID NO: 293)TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYA KKTGDQK GYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGE R NLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWL AYIQELRNAsCpf1(R912A)- Residue A912 is indicated in bold underlining andresidues 661-667 are indicated in italics and underlining.(SEQ ID NO: 294) TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYA KKTGDQK GYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGE A NLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

In some embodiments, the nucleic acid programmable DNA binding proteinis a Cpf1 protein from a Lachnospiraceae species (LbCpf1). Cpf1 proteinsform Lachnospiraceae species have been described previously have beendescribed previously and would be apparent to the skilled artisan.Exemplary Lachnospiraceae Cpf1 proteins (LbCpf1) include, withoutlimitation, any of the LbCpf1 proteins provided herein.

Wild-type LbCpf1 - Residues R836 and R1138 is indicated in boldunderlining. (SEQ ID NO: 295)MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGE R NLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQM R NSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHLbCpf1 (R836A) - Residue A836 is indicated in bold underlining.(SEQ ID NO: 296)MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGE A NLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHLbCpf1 (R1138A)- Residue A1138 is indicated in bold underlining.(SEQ ID NO: 297)MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGS SEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQM A NSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH

In some embodiments, the Cpf1 protein is a crippled Cpf1 protein. Asused herein a “crippled Cpf 1” protein is a Cpf1 protein havingdiminished nuclease activity as compared to a wild-type Cpf1 protein. Insome embodiments, the crippled Cpf1 protein preferentially cuts thetarget strand more efficiently than the non-target strand. For example,the Cpf1 protein preferentially cuts the strand of a duplexed nucleicacid molecule in which a nucleotide to be edited resides. In someembodiments, the crippled Cpf1 protein preferentially cuts thenon-target strand more efficiently than the target strand. For example,the Cpf1 protein preferentially cuts the strand of a duplexed nucleicacid molecule in which a nucleotide to be edited does not reside. Insome embodiments, the crippled Cpf1 protein preferentially cuts thetarget strand at least 5% more efficiently than it cuts the non-targetstrand. In some embodiments, the crippled Cpf1 protein preferentiallycuts the target strand at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,50%, 60%, 70%, 80%, 90%, or at least 100% more efficiently than it cutsthe non-target strand.

In some embodiments, a crippled Cpf1 protein is a non-naturallyoccurring Cpf1 protein. In some embodiments, the crippled Cpf1 proteincomprises one or more mutations relative to a wild-type Cpf1 protein. Insome embodiments, the crippled Cpf1 protein comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutationsrelative to a wild-type Cpf1 protein. In some embodiments, the crippledCpf1 protein comprises an R836A mutation mutation as set forth in SEQ IDNO: 295, or in a corresponding amino acid in another Cpf1 protein. Itshould be appreciated that a Cpf1 comprising a homologous residue (e.g.,a corresponding amino acid) to R836A of SEQ ID NO: 295 could also bemutated to achieve similar results. In some embodiments, the crippledCpf1 protein comprises a R1138A mutation as set forth in SEQ ID NO: 295,or in a corresponding amino acid in another Cpf1 protein. In someembodiments, the crippled Cpf1 protein comprises an R912A mutationmutation as set forth in SEQ ID NO: 293, or in a corresponding aminoacid in another Cpf1 protein. Without wishing to be bound by anyparticular theory, residue R838 of SEQ ID NO: 295 (LbCpf1) and residueR912 of SEQ ID NO: 293 (AsCpf1) are examples of corresponding (e.g.,homologous) residues. For example, a portion of the alignment betweenSEQ ID NO: 293 and 295 shows that R912 and R838 are correspondingresidues.

AsCpf1 YQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYIIVIDSTGKILEQRSLNTIQ--(SEQ ID NO: 298) LbCpf1KCPKN-IFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINN(SEQ ID NO: 299)    *   *:* .*.. **.. :  :**********:**.*:*..*:*:** *** *

In some embodiments, any of the Cpf1 proteins provided herein comprisesone or more amino acid deletions. In some embodiments, any of the Cpf1proteins provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions. Withoutwishing to be bound by any particular theory, there is a helical regionin Cpf1, which includes residues 661-667 of AsCpf1 (SEQ ID NO: 293),that may obstruct the function of a deaminase (e.g., APOBEC) that isfused to the Cpf1. This region comprises the amino acid sequence KKTGDQK(SEQ ID NO: 300). Accordingly, aspects of the disclosure provide Cpf1proteins comprising mutations (e.g., deletions) that disrupt thishelical region in Cpf1. In some embodiments, the Cpf1 protein comprisesone or more deletions of the following residues in SEQ ID NO: 293, orone or more corresponding deletions in another Cpf1 protein: K661, K662,T663, G664, D665, Q666, and K667. In some embodiments, the Cpf1 proteincomprises a T663 and a D665 deletion in SEQ ID NO: 293, or correspondingdeletions in another Cpf1 protein. In some embodiments, the Cpf1 proteincomprises a K662, T663, D665, and Q666 deletion in SEQ ID NO: 293, orcorresponding deletions in another Cpf1 protein. In some embodiments,the Cpf1 protein comprises a K661, K662, T663, D665, Q666 and K667deletion in SEQ ID NO: 293, or corresponding deletions in another Cpf1protein.

AsCpf1 (deleted T663 and D665) (SEQ ID NO: 301)TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKGQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNAsCpf1 (deleted K662, T663, D665, and Q666) (SEQ ID NO: 302)TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFC KYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKGKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQEL RNAsCpf1 (deleted K661, K662, T663,D665, Q666, and K667) (SEQ ID NO: 303)TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAGGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELR N 

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) is a nucleic acid programmable DNA binding protein that doesnot require a canonical (NGG) PAM sequence in the target sequence. Insome embodiments, the napDNAbp is an Argonaute protein. One example ofsuch a nucleic acid programmable DNA binding protein is an Argonauteprotein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guidedendonuclease. NgAgo binds 5′-phosphorylated ssDNA of ˜24 nucleotides(gDNA) in length to guide it to a target site and makes DNAdouble-strand breaks at the gDNA site. In contrast to Cas9, theNgAgo-gDNA system does not require a protospacer-adjacent motif (PAM).Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the basesthat may be targeted. The characterization and use of NgAgo have beendescribed in Gao et al., Nat. Biotechnol., 2016 Jul; 34(7):768-73.PubMed PMID: 27136078; Swarts et al., Nature 507(7491) (2014):258-61;and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each ofwhich is incorporated herein by reference. The sequence ofNatronobacterium gregoryi Argonaute is provided in SEQ ID NO: 304.

In some embodiments, the napDNAbp is an Argonaute protein. In someembodiments, the napDNAbp comprises an amino acid sequence that is atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to a naturally-occurringArgonaute protein. In some embodiments, the napDNAbp is anaturally-occurring Argonaute protein. In some embodiments, the napDNAbpcomprises an amino acid sequence that is at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to any one of SEQ ID NO: 304. In some embodiments, thenapDNAbp comprises an amino acid sequence of any one SEQ ID NO: 304.

Wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 304)(SEQ ID NO: 304) MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFEQDNGERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTTVENATAQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLHTYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDLDCGVRVEADETRTLDYTTAKDRLLARELVEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDEFDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGHIVWGLRDECATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDDAVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAERLDPVRLNGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAVVLPEQQADTCKAQWDTMADLLNQAGAPPTRSETVQYDAFSSPESISLNVAGAIDPSEVDAAFVVLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALGLLAAAGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGHSSTRPQLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATEFLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVATFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNSTARLPITTAYADQASTHATKGYLVQTGAFESNVGFL

In some embodiments, the napDNAbp is a prokaryotic homolog of anArgonaute protein. Prokaryotic homologs of Argonaute proteins are knownand have been described, for example, in Makarova K., et al.,“Prokaryotic homologs of Argonaute proteins are predicted to function askey components of a novel system of defense against mobile geneticelements”, Biol. Direct. 2009 Aug. 25; 4:29. doi:10.1186/1745-6150-4-29, is incorporated herein by reference. In someembodiments, the napDNAbp is a Marinitoga piezophila Argunaute (MpAgo)protein. The CRISPR-associated Marinitoga piezophila Argonaute (MpAgo)protein cleaves single-stranded target sequences using 5′-phosphorylatedguides. The 5′ guides are used by all known Argonautes. The crystalstructure of an MpAgo-RNA complex shows a guide strand binding sitecomprising residues that block 5′ phosphate interactions. This datasuggests the evolution of an Argonaute subclass with noncanonicalspecificity for a 5′-hydroxylated guide. See, e.g., Kaya et al., “Abacterial Argonaute with noncanonical guide RNA specificity”, Proc NatlAcad Sci USA. 2016 Apr. 12; 113(15):4057-62, the entire contents ofwhich are hereby incorporated by reference). It should be appreciatedthat other Argonaute proteins may be used in any of the fusion proteins(e.g., base editors) described herein, for example, to guide a deaminase(e.g., cytidine deaminase) to a target nucleic acid (e.g., ssRNA).

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) is a single effector of a microbial CRISPR-Cas system. Singleeffectors of microbial CRISPR-Cas systems include, without limitation,Cas9, Cpf1, C2c1, C2c2, and C2c3. Typically, microbial CRISPR-Cassystems are divided into Class 1 and Class 2 systems. Class 1 systemshave multisubunit effector complexes, while Class 2 systems have asingle protein effector. Cas9 and Cpf1 are Class 2 effectors. Inaddition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems(C2c1, C2c2, and C2c3) have been described by Shmakov et al., “Discoveryand Functional Characterization of Diverse Class 2 CRISPR Cas Systems”,Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which areherein incorporated by reference. Effectors of two of the systems, C2c1and C2c3, contain RuvC-like endonuclease domains related to Cpf1. Athird system, C2c2 contains an effector with two predicted HEPN RNasedomains. Production of mature CRISPR RNA is tracrRNA-independent, unlikeproduction of CRISPR RNA by C2c1. C2c1 depends on both CRISPR RNA andtracrRNA for DNA cleavage. Bacterial C2c2 has been shown to possess aunique RNase activity for CRISPR RNA maturation distinct from itsRNA-activated single-stranded RNA degradation activity. These RNasefunctions are different from each other and from the CRISPRRNA-processing behavior of Cpf1. See, e.g., East-Seletsky, et al., “Twodistinct RNase activities of CRISPR-C2c2 enable guide-RNA processing andRNA detection”, Nature, 2016 Oct. 13; 538(7624):270-273, the entirecontents of which are hereby incorporated by reference. In vitrobiochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2is guided by a single CRISPR RNA and can be programmed to cleave ssRNAtargets carrying complementary protospacers. Catalytic residues in thetwo conserved HEPN domains mediate cleavage. Mutations in the catalyticresidues generate catalytically inactive RNA-binding proteins. See e.g.,Abudayyeh et al., “C2c2 is a single-component programmable RNA-guidedRNA-targeting CRISPR effector,” Science, 2016 Aug. 5; 353(6299), theentire contents of which are hereby incorporated by reference.

The crystal structure of Alicyclobaccillus acidoterrastris C2c1(AacC2c1) has been reported in complex with a chimeric single-moleculeguide RNA (sgRNA). See, e.g., Liu et al., “C2c1-sgRNA Complex StructureReveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19;65(2):310-322, incorporated herein by reference. The crystal structurehas also been reported for Alicyclobacillus acidoterrestris C2c1 boundto target DNAs as ternary complexes. See, e.g., Yang et al.,“PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Casendonuclease”, Cell, 2016 Dec. 15; 167(7):1814-1828, the entire contentsof which are hereby incorporated by reference. Catalytically competentconformations of AacC2c1, both with target and non-target DNA strands,have been captured independently positioned within a single RuvCcatalytic pocket, with C2c1-mediated cleavage resulting in a staggeredseven-nucleotide break of target DNA. Structural comparisons betweenC2c1 ternary complexes and previously identified Cas9 and Cpf1counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9systems.

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) of any of the fusion proteins provided herein is a C2c1, aC2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2c1protein. In some embodiments, the napDNAbp is a C2c2 protein. In someembodiments, the napDNAbp is a C2c3 protein. In some embodiments, thenapDNAbp comprises an amino acid sequence that is at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least99.5% identical to a naturally-occurring C2c1, C2c2, or C2c3 protein. Insome embodiments, the napDNAbp is a naturally-occurring C2c1, C2c2, orC2c3 protein. In some embodiments, the napDNAbp comprises an amino acidsequence that is at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% identical to any one of SEQID NOs: 305-307. In some embodiments, the napDNAbp comprises an aminoacid sequence of any one SEQ ID NOs: 305-307. It should be appreciatedthat C2c1, C2c2, or C2c3 from other bacterial species may also be usedin accordance with the present disclosure.

C2c1 (uniprot.org/uniprot/T0D7A2#)sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS =Alicyclobacillus acidoterrestris (strain ATCC 49025/DSM 3922/CIP106132/NCIMB 13137/GD3B) GN = c2c1 PE = 1 SV = 1 (SEQ ID NO: 305)MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQDSACENTGDIC2c2 (uniprot.org/uniprot/P0DOC6) >sp|P0DOC6|C2C2_LEPSD CRISPR-associated endoribonuclease C2c2OS = Leptotrichia shahii (strain DSM 19757/CCUG 47503/CIP 107916/JCM 16776/LB37) GN = c2c2 PE = 1 SV = 1 (SEQ ID NO: 306)MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTLC2c3, translated from >CEPX01008730.1 marine metagenome genomeassembly TARA_037_MES_0.1-0.22, contigTARA_037_MES_0.1-0.22_scaffo1d22115_1, whole genome shotgun sequence.(SEQ ID NO: 307) MRSNYHGGRNARQWRKQISGLARRTKETVFTYKFPLETDAAEIDFDKAVQTYGIAEGVGHGSLIGLVCAFHLSGFRLFSKAGEAMAFRNRSRYPTDAFAEKLSAIMGIQLPTLSPEGLDLIFQSPPRSRDGIAPVWSENEVRNRLYTNWTGRGPANKPDEHLLEIAGEIAKQVFPKFGGWDDLASDPDKALAAADKYFQSQGDFPSIASLPAAIMLSPANSTVDFEGDYIAIDPAAETLLHQAVSRCAARLGRERPDLDQNKGPFVSSLQDALVSSQNNGLSWLFGVGFQHWKEKSPKELIDEYKVPADQHGAVTQVKSFVDAIPLNPLFDTTHYGEFRASVAGKVRSWVANYWKRLLDLKSLLATTEFTLPESISDPKAVSLFSGLLVDPQGLKKVADSLPARLVSAEEAIDRLMGVGIPTAADIAQVERVADEIGAFIGQVQQFNNQVKQKLENLQDADDEEFLKGLKIELPSGDKEPPAINRISGGAPDAAAEISELEEKLQRLLDARSEHFQTISEWAEENAVTLDPIAAMVELERLRLAERGATGDPEEYALRLLLQRIGRLANRVSPVSAGSIRELLKPVFMEEREFNLFFHNRLGSLYRSPYSTSRHQPFSIDVGKAKAIDWIAGLDQISSDIEKALSGAGEALGDQLRDWINLAGFAISQRLRGLPDTVPNALAQVRCPDDVRIPPLLAMLLEEDDIARDVCLKAFNLYVSAINGCLFGALREGFIVRTRFQRIGTDQIHYVPKDKAWEYPDRLNTAKGPINAAVSSDWIEKDGAVIKPVETVRNLSSTGFAGAGVSEYLVQAPHDWYTPLDLRDVAHLVTGLPVEKNITKLKRLTNRTAFRMVGASSFKTHLDSVLLSDKIKLGDFTIIIDQHYRQSVTYGGKVKISYEPERLQVEAAVPVVDTRDRTVPEPDTLFDHIVAIDLGERSVGFAVFDIKSCLRTGEVKPIHDNNGNPVVGTVAVPSIRRLMKAVRSHRRRRQPNQKVNQTYSTALQNYRENVIGDVCNRIDTLMERYNAFPVLEFQIKNFQAGAKQLEIVYGS 

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) of any of the fusion proteins provided herein is a Cas9 fromarchaea (e.g. nanoarchaea), which constitute a domain and kingdom ofsingle-celled prokaryotic microbes. In some embodiments, the napDNAbp isCasX or CasY, which have been described in, for example, Burstein etal., “New CRISPR—Cas systems from uncultivated microbes.” Cell Res. 2017Feb. 21. doi: 10.1038/cr.2017.21, which is incorporated herein byreference. Using genome-resolved metagenomics, a number of CRISPR—Cassystems were identified, including the first reported Cas9 in thearchaeal domain of life. This divergent Cas9 protein was found innanoarchaea as part of an active CRISPR—Cas system. In bacteria, twopreviously unknown systems were discovered, CRISPR—CasX and CRISPR—CasY, which are among the most compact systems yet discovered. In someembodiments, Cas9 refers to CasX, or a variant of CasX. In someembodiments, Cas9 refers to a CasY, or a variant of CasY. It should beappreciated that other RNA-guided DNA binding proteins may be used as anucleic acid programmable DNA binding protein (napDNAbp) and are withinthe scope of this disclosure.

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) of any of the fusion proteins provided herein is a CasX orCasY protein. In some embodiments, the napDNAbp is a CasX protein. Insome embodiments, the napDNAbp is a CasY protein. In some embodiments,the napDNAbp comprises an amino acid sequence that is at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.5% identical to a naturally-occurring CasX or CasY protein. Insome embodiments, the napDNAbp is a naturally-occurring CasX or CasYprotein. In some embodiments, the napDNAbp comprises an amino acidsequence that is at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% identical to any one of SEQID NOs: 308-310. In some embodiments, the napDNAbp comprises an aminoacid sequence of any one SEQ ID NOs: 308-310. It should be appreciatedthat CasX and CasY from other bacterial species may also be used inaccordance with the present disclosure.

CasX (uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS =Sulfolobus islandicus (strain HVE10/4) GN = SiH_0402 PE = 4 SV = 1(SEQ ID NO: 308)MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS =Sulfolobus islandicus (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1(SEQ ID NO: 309)MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEGCasY (ncbi.nlm.nih.gov/protein/APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteriagroup bacterium] (SEQ ID NO: 310)MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKNIKVLGQMKKI

Cas9 Domains of Nucleobase Editors

Some aspects of the disclosure provide fusion proteins comprising a Cas9domain. Non-limiting, exemplary Cas9 domains are provided herein. TheCas9 domain may be a nuclease active Cas9 domain, a nuclease inactiveCas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain isa nuclease active domain. For example, the Cas9 domain may be a Cas9domain that cuts both strands of a duplexed nucleic acid (e.g., bothstrands of a duplexed DNA molecule). In some embodiments, the Cas9domain comprises any one of the amino acid sequences as set forth in SEQID NOs: 6 or 11-260. In some embodiments the Cas9 domain comprises anamino acid sequence that is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to any one of the amino acid sequences set forth in SEQ IDNOs: 6 or 11-260. In some embodiments, the Cas9 domain comprises anamino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, or more mutations compared to any one of the amino acid sequencesset forth in SEQ ID NOs: 6 or 11-260. In some embodiments, the Cas9domain comprises an amino acid sequence that has at least 10, at least15, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 150, at least200, at least 250, at least 300, at least 350, at least 400, at least500, at least 600, at least 700, at least 800, at least 900, at least1000, at least 1100, or at least 1200 identical contiguous amino acidresidues as compared to any one of the amino acid sequences set forth inSEQ ID NOs: 6 or 11-260.

In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain(dCas9). For example, the dCas9 domain may bind to a duplexed nucleicacid molecule (e.g., via a gRNA molecule) without cleaving either strandof the duplexed nucleic acid molecule. In some embodiments, thenuclease-inactive dCas9 domain comprises a D10X mutation and a H840Xmutation of the amino acid sequence set forth in SEQ ID NO: 6, or acorresponding mutation in any of the amino acid sequences provided inSEQ ID NOs: 11-260, wherein X is any amino acid change. In someembodiments, the nuclease-inactive dCas9 domain comprises a D10Amutation and a H840A mutation of the amino acid sequence set forth inSEQ ID NO: 6, or a corresponding mutation in any of the amino acidsequences provided in SEQ ID NOs: 11-260. As one example, anuclease-inactive Cas9 domain comprises the amino acid sequence setforth in SEQ ID NO: 311 (Cloning vector pPlatTET-gRNA2, Accession No.BAV54124).

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ LGGD (SEQ IDNO: 311; see, e.g., Qi et al., Repurposing CRISPR as an RNA-guidedplatform for sequence-specific control of gene expression. Cell. 2013;152(5):1173-83, the entire contents of which are incorporated herein byreference).

Additional suitable nuclease-inactive dCas9 domains will be apparent tothose of skill in the art based on this disclosure and knowledge in thefield, and are within the scope of this disclosure. Such additionalexemplary suitable nuclease-inactive Cas9 domains include, but are notlimited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863Amutant domains (See, e.g., Prashant et al., CAS9 transcriptionalactivators for target specificity screening and paired nickases forcooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein byreference). In some embodiments the dCas9 domain comprises an amino acidsequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical toany one of the dCas9 domains provided herein. In some embodiments, theCas9 domain comprises an amino acid sequences that has 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any oneof the amino acid sequences set forth in SEQ ID NOs: 6 or 11-260. Insome embodiments, the Cas9 domain comprises an amino acid sequence thathas at least 10, at least 15, at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90, at least100, at least 150, at least 200, at least 250, at least 300, at least350, at least 400, at least 500, at least 600, at least 700, at least800, at least 900, at least 1000, at least 1100, or at least 1200identical contiguous amino acid residues as compared to any one of theamino acid sequences set forth in SEQ ID NOs: 6 or 11-260.

In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickasemay be a Cas9 protein that is capable of cleaving only one strand of aduplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In someembodiments the Cas9 nickase cleaves the target strand of a duplexednucleic acid molecule, meaning that the Cas9 nickase cleaves the strandthat is base paired to (complementary to) a gRNA (e.g., an sgRNA) thatis bound to the Cas9. In some embodiments, a Cas9 nickase comprises aD10A mutation and has a histidine at position 840 of SEQ ID NO: 6, or amutation in any of SEQ ID NOs: 11-260. For example, a Cas9 nickase maycomprise the amino acid sequence as set forth in SEQ ID NO: 674. In someembodiments the Cas9 nickase cleaves the non-target, non-base-editedstrand of a duplexed nucleic acid molecule, meaning that the Cas9nickase cleaves the strand that is not base paired to a gRNA (e.g., ansgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickasecomprises an H840A mutation and has an aspartic acid residue at position10 of SEQ ID NO: 6, or a corresponding mutation in any of SEQ ID NOs:11-260. In some embodiments the Cas9 nickase comprises an amino acidsequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical toany one of the Cas9 nickases provided herein. Additional suitable Cas9nickases will be apparent to those of skill in the art based on thisdisclosure and knowledge in the field, and are within the scope of thisdisclosure.

Cas9 Domains with Reduced PAM Exclusivity

Some aspects of the disclosure provide Cas9 domains that have differentPAM specificities. Typically, Cas9 proteins, such as Cas9 from S.pyogenes (spCas9), require a canonical NGG PAM sequence to bind aparticular nucleic acid region. This may limit the ability to editdesired bases within a genome. In some embodiments, the base editingfusion proteins provided herein may need to be placed at a preciselocation, for example where a target base is placed within a 4 baseregion (e.g., a “deamination window”), which is approximately 15 basesupstream of the PAM. See Komor, A. C., et al., “Programmable editing ofa target base in genomic DNA without double-stranded DNA cleavage”Nature 533, 420-424 (2016), the entire contents of which are herebyincorporated by reference. Accordingly, in some embodiments, any of thefusion proteins provided herein may contain a Cas9 domain that iscapable of binding a nucleotide sequence that does not contain acanonical (e.g., NGG) PAM sequence. Cas9 domains that bind tonon-canonical PAM sequences have been described in the art and would beapparent to the skilled artisan. For example, Cas9 domains that bindnon-canonical PAM sequences have been described in Kleinstiver, B. P.,et al., “Engineered CRISPR-Cas9 nucleases with altered PAMspecificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., etal., “Broadening the targeting range of Staphylococcus aureusCRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33,1293-1298 (2015); the entire contents of each are hereby incorporated byreference.

In some embodiments, the Cas9 domain is a Cas9 domain fromStaphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domainis a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or aSaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises theamino acid sequence SEQ ID NO: 313. In some embodiments, the SaCas9comprises a N579X mutation of SEQ ID NO: 313, or a correspondingmutation in any of the amino acid sequences provided in SEQ ID NOs:11-260, wherein X is any amino acid except for N. In some embodiments,the SaCas9 comprises a N579A mutation of SEQ ID NO: 313, or acorresponding mutation in any of the amino acid sequences provided inSEQ ID NOs: 11-260. In some embodiments, the SaCas9 domain, the SaCas9ddomain, or the SaCas9n domain can bind to a nucleic acid seuqnce havinga non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9ddomain, or the SaCas9n domain can bind to a nucleic acid sequence havinga NNGRRT (SEQ ID NO: 312) PAM sequence. In some embodiments, the SaCas9domain comprises one or more of a E781X, a N967X, and a R1014X mutationof SEQ ID NO: 313, or a corresponding mutation in any of the amino acidsequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid.In some embodiments, the SaCas9 domain comprises one or more of a E781K,a N967K, and a R1014H mutation of SEQ ID NO: 313, or one or morecorresponding mutation in any of the amino acid sequences provided inSEQ ID NOs: 11-260. In some embodiments, the SaCas9 domain comprises aE781K, a N967K, or a R1014H mutation of SEQ ID NO: 313, or correspondingmutations in any of the amino acid sequences provided in SEQ ID NOs:11-260.

In some embodiments, the Cas9 domain of any of the fusion proteinsprovided herein comprises an amino acid sequence that is at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to any one of SEQ ID NOs:313-315. In some embodiments, the Cas9 domain of any of the fusionproteins provided herein comprises the amino acid sequence of any one ofSEQ ID NOs: 313-315. In some embodiments, the Cas9 domain of any of thefusion proteins provided herein consists of the amino acid sequence ofany one of SEQ ID NOs: 313-315.

Exemplary SaCas9 sequence (SEQ ID NO: 313)KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE N SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKS KKHPQII KKG Residue N579 of SEQ ID NO: 313, which is underlined and in bold, maybe mutated (e.g., to a A579) to yield a SaCas9 nickase.Exemplary SaCas9n sequence (SEQ ID NO: 314)KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII KKG.Residue A579 of SEQ ID NO: 314, which can be mutated from N579 ofSEQ ID NO: 314 to yield a SaCas9 nickase, is underlined and in bold.Exemplary SaKKH Cas9 (SEQ ID NO: 315)KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNR K LINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFY K NDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPP H IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII KKG.Residue A579 of SEQ ID NO: 315, which can be mutated from N579 ofSEQ ID NO: 315 to yield a SaCas9 nickase, is underlined and in bold.Residues K781, K967, and H1014 of SEQ ID NO: 315, which can bemutated from E781, N967, and R1014 of SEQ ID NO: 315 to yield aSaKKH Cas9 are underlined and in italics.

In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcuspyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nucleaseactive SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase(SpCas9n). In some embodiments, the SpCas9 comprises the amino acidsequence SEQ ID NO: 316. In some embodiments, the SpCas9 comprises a D9Xmutation of SEQ ID NO: 316, or a corresponding mutation in any of theamino acid sequences provided in SEQ ID NOs: 11-260, wherein X is anyamino acid except for D. In some embodiments, the SpCas9 comprises a D9Amutation of SEQ ID NO: 316, or a corresponding mutation in any of theamino acid sequences provided in SEQ ID NOs: 11-260. In someembodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9ndomain can bind to a nucleic acid seuqnce having a non-canonical PAM. Insome embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9ndomain can bind to a nucleic acid sequence having a NGG, a NGA, or aNGCG PAM sequence. In some embodiments, the SpCas9 domain comprises oneor more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 316,or a corresponding mutation in any of the amino acid sequences providedin SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments,the SpCas9 domain comprises one or more of a D1134E, R1334Q, and T1336Rmutation of SEQ ID NO: 316, or a corresponding mutation in any of theamino acid sequences provided in SEQ ID NOs: 11-260. In someembodiments, the SpCas9 domain comprises a D1134E, a R1334Q, and aT1336R mutation of SEQ ID NO: 316, or corresponding mutations in any ofthe amino acid sequences provided in SEQ ID NOs: 11-260. In someembodiments, the SpCas9 domain comprises one or more of a D1134X, aR1334X, and a T1336X mutation of SEQ ID NO: 316, or a correspondingmutation in any of the amino acid sequences provided in SEQ ID NOs:11-260, wherein X is any amino acid. In some embodiments, the SpCas9domain comprises one or more of a D1134V, a R1334Q, and a T1336Rmutation of SEQ ID NO: 316, or a corresponding mutation in any of theamino acid sequences provided in SEQ ID NOs: 11-260. In someembodiments, the SpCas9 domain comprises a D1134V, a R1334Q, and aT1336R mutation of SEQ ID NO: 316, or corresponding mutations in any ofthe amino acid sequences provided in SEQ ID NOs: 11-260. In someembodiments, the SpCas9 domain comprises one or more of a D1134X, aG1217X, a R1334X, and a T1336X mutation of SEQ ID NO: 316, or acorresponding mutation in any of the amino acid sequences provided inSEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments,the SpCas9 domain comprises one or more of a D1134V, a G1217R, a R1334Q,and a T1336R mutation of SEQ ID NO: 316, or a corresponding mutation inany of the amino acid sequences provided in SEQ ID NOs: 11-260. In someembodiments, the SpCas9 domain comprises a D1134V, a G1217R, a R1334Q,and a T1336R mutation of SEQ ID NO: 316, or corresponding mutations inany of the amino acid sequences provided in SEQ ID NOs: 11-260.

In some embodiments, the Cas9 domain of any of the fusion proteinsprovided herein comprises an amino acid sequence that is at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to any one of SEQ ID NOs:316-320. In some embodiments, the Cas9 domain of any of the fusionproteins provided herein comprises the amino acid sequence of any one ofSEQ ID NOs: 316-320. In some embodiments, the Cas9 domain of any of thefusion proteins provided herein consists of the amino acid sequence ofany one of SEQ ID NOs: 316-320.

Exemplary SpCas9 (SEQ ID NO: 316)DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Exemplary SpCas9n (SEQ ID NO: 317)DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Exemplary SpEQR Cas9 (SEQ ID NO: 318)DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF E SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGDResidues E1134, Q1334, and R1336 of SEQ ID NO: 318, which canbe mutated from D1134, R1334, and T1336 of SEQ ID NO: 316 toyield a SpEQR Cas9, are underlined and in bold. Exemplary SpVQR Cas9(SEQ ID NO: 319)DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGDResidues V1134, Q1334, and R1336 of SEQ ID NO: 319, which canbe mutated from D1134, R1334, and T1336 of SEQ ID NO: 316 toyield a SpVQR Cas9, are underlined and in bold. Exemplary SpVRER Cas9(SEQ ID NO: 320)DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQSITGLYETRIDLSQLGGDResidues V1134, R1217, Q1334, and R1336 of SEQ ID NO: 320,which can be mutated from D1134, G1217, R1334, and T1336 of SEQID NO: 316 to yield a SpVRER Cas9, are underlined and in bold.

High Fidelity Base Editors

Some aspects of the disclosure provide Cas9 fusion proteins (e.g., anyof the fusion proteins provided herein) comprising a Cas9 domain thathas high fidelity. Additional aspects of the disclosure provide Cas9fusion proteins (e.g., any of the fusion proteins provided herein)comprising a Cas9 domain with decreased electrostatic interactionsbetween the Cas9 domain and a sugar-phosphate backbone of a DNA, ascompared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain(e.g., a wild type Cas9 domain) comprises one or more mutations thatdecreases the association between the Cas9 domain and a sugar-phosphatebackbone of a DNA. In some embodiments, any of the Cas9 fusion proteinsprovided herein comprise one or more of a N497X, a R661X, a Q695X,and/or a Q926X mutation of the amino acid sequence provided in SEQ IDNO: 6, or a corresponding mutation in any of the amino acid sequencesprovided in SEQ ID NOs: 11-260, wherein X is any amino acid. In someembodiments, any of the Cas9 fusion proteins provided herein compriseone or more of a N497A, a R661A, a Q695A, and/or a Q926A mutation of theamino acid sequence provided in SEQ ID NO: 10, or a correspondingmutation in any of the amino acid sequences provided in SEQ ID NOs:11-260. In some embodiments, the Cas9 domain comprises a D10A mutationof the amino acid sequence provided in SEQ ID NO: 6, or a correspondingmutation in any of the amino acid sequences provided in SEQ ID NOs:11-260. In some embodiments, the Cas9 domain (e.g., of any of the fusionproteins provided herein) comprises the amino acid sequence as set forthin SEQ ID NO: 321. In some embodiments, the fusion protein comprises theamino acid sequence as set forth in SEQ ID NO: 322. Cas9 domains withhigh fidelity are known in the art and would be apparent to the skilledartisan. For example, Cas9 domains with high fidelity have beendescribed in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9nucleases with no detectable genome-wide off-target effects.” Nature529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineeredCas9 nucleases with improved specificity.” Science 351, 84-88 (2015);the entire contents of each are incorporated herein by reference.

It should be appreciated that the base editors provided herein, forexample, base editor 2 (BE2) or base editor 3 (BE3), may be convertedinto high fidelity base editors by modifying the Cas9 domain asdescribed herein to generate high fidelity base editors, for example,high fidelity base editor 2 (HF-BE2) or high fidelity base editor 3(HF-BE3). In some embodiments, base editor 2 (BE2) comprises a deaminasedomain, a dCas9, and a UGI domain. In some embodiments, base editor 3(BE3) comprises a deaminase domain, anCas9 domain and a UGI domain.

Cas9 domain where mutations relative to Cas9 of SEQ ID NO: 10are shown in bold and underlines (SEQ ID NO: 321) DKKYSIGL AIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM T AFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ LVETR AITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD HF-BE3 (SEQ ID NO: 322)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

Deaminase Domains of the Fusion Proteins

Some aspects of the disclosure provide fusion proteins comprising one ormore nucleic acid editing domains (e.g., deaminase domains). In someembodiments, the nucleic acid editing domain can catalyze a C to U basechange. In some embodiments, the nucleic acid editing domain is adeaminase domain. In some embodiments, the deaminase is a cytidinedeaminase or a cytidine deaminase. In some embodiments, the deaminase isan apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. Insome embodiments, the deaminase is an APOBEC1 deaminase. In someembodiments, the deaminase is an APOBEC2 deaminase. In some embodiments,the deaminase is an APOBEC3 deaminase. In some embodiments, thedeaminase is an APOBEC3A deaminase. In some embodiments, the deaminaseis an APOBEC3B deaminase. In some embodiments, the deaminase is anAPOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3Ddeaminase. In some embodiments, the deaminase is an APOBEC3E deaminase.In some embodiments, the deaminase is an APOBEC3F deaminase. In someembodiments, the deaminase is an APOBEC3G deaminase. In someembodiments, the deaminase is an APOBEC3H deaminase. In someembodiments, the deaminase is an APOBEC4 deaminase. In some embodiments,the deaminase is an activation-induced deaminase (AID). In someembodiments, the deaminase is a vertebrate deaminase. In someembodiments, the deaminase is an invertebrate deaminase. In someembodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow,dog, rat, or mouse deaminase. In some embodiments, the deaminase is ahuman deaminase. In some embodiments, the deaminase is a rat deaminase,e.g., rAPOBEC1. In some embodiments, the deaminase is a Petromyzonmarinus cytidine deaminase 1 (pmCDA1). In some embodiments, thedeaminase is a human APOBEC3G (SEQ ID NO: 333). In some embodiments, thedeaminase is a fragment of the human APOBEC3G (SEQ ID NO: 356). In someembodiments, the deaminase is a human APOBEC3G variant comprising aD316R_D317R mutation (SEQ ID NO: 355). In some embodiments, thedeaminase is a fragment of the human APOBEC3G and comprising mutationscorresponding to the D316R_D317R mutations in SEQ ID NO: 333 (SEQ ID NO:357).

In some embodiments, the nucleic acid editing domain is at least 80%, atleast 85%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical toany of the deaminase domains provided herein. In some embodiments, thenucleic acid editing domain is at least 80%, at least 85%, at least 90%,at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to the deaminase domain of anyone of SEQ ID NOs: 323-361. In some embodiments, the nucleic acidediting domain comprises the amino acid sequence of any one of SEQ IDNOs: 323-361.

Deaminase Domains that Modulate the Editing Window of Base Editors

Some aspects of the disclosure are based on the recognition thatmodulating the deaminase domain catalytic activity of any of the fusionproteins provided herein, for example by making point mutations in thedeaminase domain, affect the processivity of the fusion proteins (e.g.,base editors). For example, mutations that reduce, but do not eliminate,the catalytic activity of a deaminase domain within a base editingfusion protein can make it less likely that the deaminase domain willcatalyze the deamination of a residue adjacent to a target residue,thereby narrowing the deamination window. The ability to narrow thedeamination window may prevent unwanted deamination of residues adjacentof specific target residues, which may decrease or prevent off-targeteffects.

In some embodiments, any of the fusion proteins provided hereincomprises a deaminase domain (e.g., a cytidine deaminase domain) thathas reduced catalytic deaminase activity. In some embodiments, any ofthe fusion proteins provided herein comprises a deaminase domain (e.g.,a cytidine deaminase domain) that has a reduced catalytic deaminaseactivity as compared to an appropriate control. For example, theappropriate control may be the deaminase activity of the deaminase priorto introducing one or more mutations into the deaminase. In otherembodiments, the appropriate control may be a wild-type deaminase. Insome embodiments, the appropriate control is a wild-type apolipoproteinB mRNA-editing complex (APOBEC) family deaminase. In some embodiments,the appropriate control is an APOBEC1 deaminase, an APOBEC2 deaminase,an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, anAPOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, or anAPOBEC3H deaminase. In some embodiments, the appropriate control is anactivation induced deaminase (AID). In some embodiments, the appropriatecontrol is a cytidine deaminase 1 from Petromyzon marinus (pmCDA1). Insome embodiments, the deaminase domain may be a deaminase domain thathas at least 1%, at least 5%, at least 15%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95% less catalytic deaminaseactivity as compared to an appropriate control.

In some embodiments, any of the fusion proteins provided herein comprisean

APOBEC deaminase comprising one or more mutations selected from thegroup consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, andR132X of rAPOBEC1 (SEQ ID NO: 349), or one or more correspondingmutations in another APOBEC deaminase, wherein X is any amino acid. Insome embodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising one or more mutations selected from thegroup consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, andR132E of rAPOBEC1 (SEQ ID NO: 349), or one or more correspondingmutations in another APOBEC deaminase.

In some embodiments, any of the fusion proteins provided herein comprisean APOBEC deaminase comprising one or more mutations selected from thegroup consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X,R326X of hAPOBEC3G (SEQ ID NO: 333), or one or more correspondingmutations in another APOBEC deaminase, wherein X is any amino acid. Insome embodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising one or more mutations selected from thegroup consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y,R326E of hAPOBEC3G (SEQ ID NO: 333), or one or more correspondingmutations in another APOBEC deaminase.

In some embodiments, any of the fusion proteins provided herein comprisean APOBEC deaminase comprising a H121R and a H122R mutation of rAPOBEC1(SEQ ID NO: 349), or one or more corresponding mutations in anotherAPOBEC deaminase. In some embodiments, any of the fusion proteinsprovided herein comprise an APOBEC deaminase comprising a R126A mutationof rAPOBEC1 (SEQ ID NO: 349), or one or more corresponding mutations inanother APOBEC deaminase. In some embodiments, any of the fusionproteins provided herein comprise an APOBEC deaminase comprising a R126Emutation of rAPOBEC1 (SEQ ID NO: 349), or one or more correspondingmutations in another APOBEC deaminase. In some embodiments, any of thefusion proteins provided herein comprise an APOBEC deaminase comprisinga R118A mutation of rAPOBEC1 (SEQ ID NO: 349), or one or morecorresponding mutations in another APOBEC deaminase. In someembodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising a W90A mutation of rAPOBEC1 (SEQ ID NO:349), or one or more corresponding mutations in another APOBECdeaminase. In some embodiments, any of the fusion proteins providedherein comprise an APOBEC deaminase comprising a W90Y mutation ofrAPOBEC1 (SEQ ID NO: 349), or one or more corresponding mutations inanother APOBEC deaminase. In some embodiments, any of the fusionproteins provided herein comprise an APOBEC deaminase comprising a R132Emutation of rAPOBEC1 (SEQ ID NO: 349), or one or more correspondingmutations in another APOBEC deaminase. In some embodiments, any of thefusion proteins provided herein comprise an APOBEC deaminase comprisinga W90Y and a R126E mutation of rAPOBEC1 (SEQ ID NO: 349), or one or morecorresponding mutations in another APOBEC deaminase. In someembodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising a R126E and a R132E mutation of rAPOBEC1(SEQ ID NO: 349), or one or more corresponding mutations in anotherAPOBEC deaminase. In some embodiments, any of the fusion proteinsprovided herein comprise an APOBEC deaminase comprising a W90Y and aR132E mutation of rAPOBEC1 (SEQ ID NO: 349), or one or morecorresponding mutations in another APOBEC deaminase. In someembodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising a W90Y, R126E, and R132E mutation ofrAPOBEC1 (SEQ ID NO: 349), or one or more corresponding mutations inanother APOBEC deaminase.

In some embodiments, any of the fusion proteins provided herein comprisean APOBEC deaminase comprising a D316R and a D317R mutation of hAPOBEC3G(SEQ ID NO: 333), or one or more corresponding mutations in anotherAPOBEC deaminase. In some embodiments, any of the fusion proteinsprovided herein comprise an APOBEC deaminase comprising a R320A mutationof hAPOBEC3G (SEQ ID NO: 333), or one or more corresponding mutations inanother APOBEC deaminase. In some embodiments, any of the fusionproteins provided herein comprise an APOBEC deaminase comprising a R320Emutation of hAPOBEC3G (SEQ ID NO: 333), or one or more correspondingmutations in another APOBEC deaminase. In some embodiments, any of thefusion proteins provided herein comprise an APOBEC deaminase comprisinga R313A mutation of hAPOBEC3G (SEQ ID NO: 333), or one or morecorresponding mutations in another APOBEC deaminase. In someembodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising a W285A mutation of hAPOBEC3G (SEQ ID NO:333), or one or more corresponding mutations in another APOBECdeaminase. In some embodiments, any of the fusion proteins providedherein comprise an APOBEC deaminase comprising a W285Y mutation ofhAPOBEC3G (SEQ ID NO: 333), or one or more corresponding mutations inanother APOBEC deaminase. In some embodiments, any of the fusionproteins provided herein comprise an APOBEC deaminase comprising a R326Emutation of hAPOBEC3G (SEQ ID NO: 333), or one or more correspondingmutations in another APOBEC deaminase. In some embodiments, any of thefusion proteins provided herein comprise an APOBEC deaminase comprisinga W285Y and a R320E mutation of hAPOBEC3G (SEQ ID NO: 333), or one ormore corresponding mutations in another APOBEC deaminase. In someembodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising a R320E and a R326E mutation of hAPOBEC3G(SEQ ID NO: 333), or one or more corresponding mutations in anotherAPOBEC deaminase. In some embodiments, any of the fusion proteinsprovided herein comprise an APOBEC deaminase comprising a W285Y and aR326E mutation of hAPOBEC3G (SEQ ID NO: 333), or one or morecorresponding mutations in another APOBEC deaminase. In someembodiments, any of the fusion proteins provided herein comprise anAPOBEC deaminase comprising a W285Y, R320E, and R326E mutation ofhAPOBEC3G (SEQ ID NO: 333), or one or more corresponding mutations inanother APOBEC deaminase.

Some exemplary suitable nucleic-acid editing domains, e.g., deaminasesand deaminase domains that can be fused to Cas9 domains according toaspects of this disclosure are provided below. It should be understoodthat, in some embodiments, the active domain of the respective sequencecan be used, e.g., the domain without a localizing signal (nuclearlocalization sequence, without nuclear export signal, cytoplasmiclocalizing signal).

Human AID: (SEQ ID NO: 323)MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(underline: nuclear localization sequence; double underline:nuclear export signal) Mouse AID: (SEQ ID NO: 324)MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDAFRMLGF(underline: nuclear localization sequence; double underline:nuclear export signal) Dog AID: (SEQ ID NO: 325)MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(underline: nuclear localization sequence; double underline:nuclear export signal) Bovine AID: (SEQ ID NO: 326)MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(underline: nuclear localization sequence; double underline:nuclear export signal) Rat AID (SEQ ID NO: 327)MAVGSKPKAALVGPHWERERIWCFLCSTGLGTQQTGQTSRWLRPAATQDPVSPPRSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYFYCWNTFVENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL(underline: nuclear localization sequence; double underline:nuclear export signal) Mouse APOBEC-3: (SEQ ID NO: 328)MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS(italic: nucleic acid editing domain) Rat APOBEC-3: (SEQ ID NO: 329)MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS(italic: nucleic acid editing domain) Rhesus macaque APOBEC-3G:(SEQ ID NO: 330)MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPE MRFLRWFHKWRQLHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGRHAELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQ ALSGRLRAI(italic: nucleic acid editing domain; underline: cytoplasmiclocalization signal) Chimpanzee APOBEC-3G: (SEQ ID NO: 331)MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSKLKYHPEMRFFHWFSKWRKLHRDQEYEVIWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDWPFWKLDLHQDYRVTCFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN(italic: nucleic acid editing domain; underline: cytoplasmiclocalization signal) Green monkey APOBEC-3G: (SEQ ID NO: 332)MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAKDHPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGRHAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI(italic: nucleic acid editing domain; underline: cytoplasmiclocalization signal) Human APOBEC-3G: (SEQ ID NO: 333)MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDWPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCFQPWDGLDEHSQDLSGRLRAILQNQEN(italic: nucleic acid editing domain; underline: cytoplasmiclocalization signal) Human APOBEC-3F: (SEQ ID NO: 334)MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFL FLDSKLQEILE(italic: nucleic acid editing domain) Human APOBEC-3B: (SEQ ID NO: 335)MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (italic: nucleic acid editing domain)Rat APOBEC-3B: (SEQ ID NO: 336)MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFYWRKKFQKGLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL Bovine APOBEC-3B:(SEQ ID NO: 337)DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAEIRFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI Chimpanzee APOBEC-3B:(SEQ ID NO: 338)MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGPCLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQPPCSSRIRETEGWASVSKE GRDLGHuman APOBEC-3C: (SEQ ID NO: 339)MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLL KRRLRESLQ(italic: nucleic acid editing domain) Gorilla APOBEC3C (SEQ ID NO: 340)MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWFCDDILSPNTNYQVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFQDTDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFKPWKGLKYNFRFLKRRLQEILE (italic: nucleic acid editing domain)Human APOBEC-3A: (SEQ ID NO: 341)MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVIWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (italic: nucleic acid editing domain)Rhesus macaque APOBEC-3A: (SEQ ID NO: 342)MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGFLCNKAKNVPCGDYGCHVELRFLCEVPSWQLDPAQTYRVTWFISWSPCFRRGCAGQVRVFLQENKHVRLRIFAARIYDYDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAILQNQGN (italic: nucleic acid editing domain)Bovine APOBEC-3A: (SEQ ID NO: 343)MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPCHAELYFLGKIHSWNLDRNQHYRLTCFISWSPCYDCAQKLTTFLKENHHISLHILASRIYTHNRFGCHQSGLCELQAAGARITIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKS QALCTELQAILKTQQN(italic: nucleic acid editing domain) Human APOBEC-3H: (SEQ ID NO: 344)MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV (italic: nucleic acid editing domain)Rhesus macaque APOBEC-3H: (SEQ ID NO: 345)MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR Human APOBEC-3D: (SEQ ID NO: 346)MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (italic: nucleic acid editing domain)Human APOBEC-1: (SEQ ID NO: 347)MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR Mouse APOBEC-1:(SEQ ID NO: 348)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK Rat APOBEC-1:(SEQ ID NO: 349)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK Human APOBEC-2:(SEQ ID NO: 350)MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK Mouse APOBEC-2:(SEQ ID NO: 351)MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Rat APOBEC-2:(SEQ ID NO: 352)MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Bovine APOBEC-2:(SEQ ID NO: 353)MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILKPetromyzon marinus CDA1 (pmCDA1) (SEQ ID NO: 353)MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV Human APOBEC3G D316R_D317R (SEQ ID NO: 355)MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN Human APOBEC3G chain A (SEQ ID NO: 356)MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRA ILQHuman APOBEC3G chain A D120R_D121R (SEQ ID NO: 357)MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRA ILQHuman AID (hAID): (SEQ ID NO: 358MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLDHuman AID-DC (hAID-DC, truncated version of hAID with 7-foldincreased activity): (SEQ ID NO: 359)MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLRat APOBEC1 (rAPOBEC1): (SEQ ID NO: 349)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK Human APOBEC1 (hAPOBEC1)(SEQ ID NO: 360)MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWRPetromyzon marinus (Lamprey) CDA1 (pmCDA1): (SEQ ID NO: 354)MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV Human APOBEC3G (hAPOBEC3G): (SEQ ID NO: 361)MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN

In some embodiments, fusion proteins as provided herein comprise thefull-length amino acid of a nucleic acid editing enzyme, e.g., one ofthe sequences provided herein. In other embodiments, however, fusionproteins as provided herein do not comprise a full-length sequence of anucleic acid editing enzyme, but only a fragment thereof. For example,in some embodiments, a fusion protein provided herein comprises afragment of a nucleic acid editing enzyme. Exemplary nucleic acidediting domains (e.g. cytidine deaminases) are provided herein, andadditional suitable sequences of such domains will be apparent to thoseof skill in the art.

Additional suitable nucleic-acid editing enzyme sequences, e.g.,deaminase enzyme and domain sequences, that can be used according toaspects of this invention, e.g., that can be fused to anuclease-inactive Cas9 domain, will be apparent to those of skill in theart based on this disclosure. In some embodiments, such additionalenzyme sequences include deaminase enzyme or deaminase domain sequencesthat are at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% similar to any of the sequences provided herein.

Uracil Glycosylase Inhibitor Fusion Proteins

Some aspects of the disclosure relate to fusion proteins that comprise auracil glycosylase inhibitor (UGI) domain. In some embodiments, thefusion protein comprises a nucleic acid programmable DNA bindingprotein, a cytidine deaminase domain, a Gam protein and a UGI domain. Insome embodiments, any of the fusion proteins provided herein thatcomprise a nucleic acid programmable DNA binding protein (e.g., a Cas9domain), a cytidine deaminase, and a Gam protein may be further fused toa UGI domain either directly or via a linker. Without wishing to bebound by any particular theory, cellular DNA-repair response to thepresence of U:G heteroduplex DNA may be responsible for the decrease innucleobase editing efficiency in cells. For example, uracil DNAglycosylase (UDG) catalyzes removal of U from DNA in cells, which mayinitiate base excision repair, with reversion of the U:G pair to a C:Gpair as the most common outcome. As demonstrated in the Examples below,Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity.Thus, this disclosure contemplates a fusion protein comprising anapDNAbp, a cytidine deaminase domain, and a Gam protein, further fusedto a UGI domain. This disclosure also contemplates a fusion proteincomprising a Cas9 nickase-nucleic acid editing domain fused to acytidine deaminase, and a Gam protein, which is further fused to a UGIdomain. It should be understood that the use of a UGI domain mayincrease the editing efficiency of a nucleic acid editing domain that iscapable of catalyzing a C to U change. For example, fusion proteinscomprising a UGI domain may be more efficient in deaminating C residues.

In some embodiments, a UGI domain comprises a wild-type UGI or a UGI asset forth in SEQ ID NO: 362. In some embodiments, the UGI proteinsprovided herein include fragments of UGI and proteins homologous to aUGI or a UGI fragment. For example, in some embodiments, a UGI domaincomprises a fragment of the amino acid sequence set forth in SEQ ID NO:362. In some embodiments, a UGI fragment comprises an amino acidsequence that comprises at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%of the amino acid sequence of any of the UGI domains provided herein. Insome embodiments, a UGI fragment comprises an amino acid sequence thatcomprises at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% of the aminoacid sequence as set forth in SEQ ID NO: 362. In some embodiments, a UGIcomprises an amino acid sequence homologous to the amino acid sequenceset forth in SEQ ID NO: 362 or an amino acid sequence homologous to afragment of the amino acid sequence set forth in SEQ ID NO: 362. In someembodiments, proteins comprising UGI or fragments of UGI or homologs ofUGI or UGI fragments are referred to as “UGI variants.” A UGI variantshares homology to UGI, or a fragment thereof. For example a UGI variantis at least 70% identical, at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical, at least 99.5% identical, or at least99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO:362. In some embodiments, the UGI variant comprises a fragment of UGI,such that the fragment is at least 70% identical, at least 80%identical, at least 90% identical, at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, at least 99%identical, at least 99.5% identical, or at least 99.9% to thecorresponding fragment of wild-type UGI or a UGI as set forth in SEQ IDNO: 362. In some embodiments, the UGI comprises the following amino acidsequence:

>sp|P14739|UNGI_BPPB2 Uracil-DNA glycosylase inhibitor (SEQ ID NO: 362)MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

Suitable UGI protein and nucleotide sequences are provided herein andadditional suitable UGI sequences are known to those in the art, andinclude, for example, those published in Wang et al., Uracil-DNAglycosylase inhibitor gene of bacteriophage PBS2 encodes a bindingprotein specific for uracil-DNA glycosylase. J. Biol. Chem.264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis andcharacterization of uracil-DNA glycosylase inhibitor protein. Role ofspecific carboxylic amino acids in complex formation with Escherichiacoli uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997);Ravishankar et al., X-ray analysis of a complex of Escherichia coliuracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. Thestructure elucidation of a prokaryotic UDG. Nucleic Acids Res.26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA fromcrystal structures of the uracil-DNA glycosylase inhibitor protein andits complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol.287:331-346(1999), the entire contents of each are incorporated hereinby reference.

It should be appreciated that additional proteins may be uracilglycosylase inhibitors. For example, other proteins that are capable ofinhibiting (e.g., sterically blocking) a uracil-DNA glycosylasebase-excision repair enzyme are within the scope of this disclosure.Additionally, any proteins that block or inhibit base-excision repair asalso within the scope of this disclosure. In some embodiments, a proteinthat binds DNA is used. In another embodiment, a substitute for UGI isused. In some embodiments, a uracil glycosylase inhibitor is a proteinthat binds single-stranded DNA. For example, a uracil glycosylaseinhibitor may be a Erwinia tasmaniensis single-stranded binding protein.In some embodiments, the single-stranded binding protein comprises theamino acid sequence (SEQ ID NO: 363). In some embodiments, a uracilglycosylase inhibitor is a protein that binds uracil. In someembodiments, a uracil glycosylase inhibitor is a protein that bindsuracil in DNA. In some embodiments, a uracil glycosylase inhibitor is acatalytically inactive uracil DNA-glycosylase protein. In someembodiments, a uracil glycosylase inhibitor is a catalytically inactiveuracil DNA-glycosylase protein that does not excise uracil from the DNA.For example, a uracil glycosylase inhibitor is a UdgX. In someembodiments, the UdgX comprises the amino acid sequence (SEQ ID NO:364). As another example, a uracil glycosylase inhibitor is acatalytically inactive UDG. In some embodiments, a catalyticallyinactive UDG comprises the amino acid sequence (SEQ ID NO: 365). Itshould be appreciated that other uracil glycosylase inhibitors would beapparent to the skilled artisan and are within the scope of thisdisclosure. In some embodiments, a uracil glycosylase inhibitor is aprotein that is homologous to any one of SEQ ID NOs: 363-365 or 366-376.In some embodiments, a uracil glycosylase inhibitor is a protein that isat least 50% identical, at least 55% identical at least 60% identical,at least 65% identical, at least 70% identical, at least 75% identical,at least 80% identical at least 85% identical, at least 90% identical,at least 95% identical, at least 96% identical, at least 98% identical,at least 99% identical, or at least 99.5% identical to any one of SEQ IDNOs: 363-365 or 366-376.

Erwinia tasmaniensis SSB (themostable single-stranded DNA binding protein) (SEQ ID NO: 363)MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETKEKTEWHRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTTEVVVNVGGTMQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGGAQQQARPQQQPQQNNAPANNEPPIDFDDDIPUdgX (binds to Uracil in DNA but does not excise) (SEQ ID NO: 364)MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARIMMIGEQPGDKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTRAAGGKRRIHKTPSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGNDFRVTQHRGEVLHVDDVPGDPALVATVHPSSLLRGPKEERESAFAGLVDD LRVAADVRPUDG (catalytically inactive human UDG, binds toUracil in DNA but does not excise) (SEQ ID NO: 365)MIGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKKAPAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKKHLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVILGQEPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGDLSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLVFLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELL QKSGKKPIDWKEL

Additional single-stranded DNA binding proteins that can be used as aUGI are shown below. It should be appreciated that other single-strandedbinding proteins may be used as a UGI, for example those described inDickey T H, Altschuler S E, Wuttke D S. Single-stranded DNA-bindingproteins:multiple domains for multiple functions. Structure. 2013 Jul.2; 21(7):1074-84.

doi: 10.1016/j.str.2013.05.013. Review.; Marceau A H. Functions ofsingle-strand DNA-binding proteins in DNA replication, recombination,and repair. Methods Mol Biol. 2012; 922:1-21. doi:10.1007/978-1-62703-032-8_1; Mijakovic, Ivan, et al; Bacterialsingle-stranded DNA-binding proteins are phosphorylated on tyrosine.Nucleic Acids Res 2006; 34 (5): 1588-1596. doi: 10.1093/nar/gkj514;Mumtsidu E, Makhov A M, Konarev P V, Svergun D I, Griffith J D, Tucker PA. Structural features of the single-stranded DNA-binding protein ofEpstein-Barrvirus. J Struct Biol. 2008 February; 161(2):172-87. Epub2007 Nov. 1; Nowak M, Olszewski M, Śpibida M, Kur J. Characterization ofsingle-strandedDNA-binding proteins from the psychrophilic bacteriaDesulfotalea psychrophila,Flavobacterium psychrophilum, Psychrobacterarcticus, Psychrobactercryohalolentis, Psychromonas ingrahamii,Psychroflexus torquis, and Photobacterium profundum. BMC Microbiol. 2014Apr. 14; 14:91. doi: 10.1186/1471-2180-14-91; Tone T, Takeuchi A, Makino0. Single-stranded DNA binding protein Gp5 of Bacillus subtilis phageΦ29 is required for viral DNA replication in growth-temperaturedependent fashion. Biosci Biotechnol Biochem. 2012; 76(12):2351-3. Epub2012 Dec. 7; Wold. REPLICATION PROTEIN A:A Heterotrimeric,Single-Stranded DNA-Binding Protein Required for Eukaryotic DNAMetabolism. Annual Review of Biochem. 1997; 66:61-92. doi:10.1146/annurev.biochem.66.1.61; Wu Y, Lu J, Kang T. Humansingle-stranded DNA binding proteins: guardians of genome stability.Acta Biochim Biophys Sin (Shanghai). 2016 July; 48(7):671-7. doi:10.1093/abbs/gmw044. Epub 2016 May 23. Review; the entire contents ofeach are hereby incorporated by reference.

mtSSB - SSBP1 single stranded DNA binding protein 1 [Homosapiens (human)] (UniProtKB: Q04837; NP_001243439.1) (SEQ ID NO: 366)MFRRPVLQVLRQFVRHESETTTSLVLERSLNRVHLLGRVGQDPVLRQVEGKNPVTIFSLATNEMWRSGDSEVYQLGDVSQKTTWHRISVFRPGLRDVAYQYVKKGSRIYLEGKIDYGEYMDKNNVRRQATTIIADNIIFLSDQTKEKESingle-stranded DNA-binding protein 3 isoform A [Mus musculus](UniProtKB - Q9D032-1; NCBI Ref: NP_076161.2) (SEQ ID NO: 367)MFAKGKGSAVPSDGQAREKLALYVYEYLLHVGAQKSAQTFLSEIRWEKNITLGEPPGFLHSWWCVFWDLYCAAPERRDTCEHSSEAKAFHDYSAAAAPSPVLGNIPPNDGMPGGPIPPGFFQGPPGSQPSPHAQPPPHNPSSMMGPHSQPFMSPRYAGGPRPPIRMGNQPPGGVPGTQPLLPNSMDPTRQQGHPNMGGSMQRMNPPRGMGPMGPGPQNYGSGMRPPPNSLGPAMPGINMGPGAGRPWPNPNSANSIPYSSSSPGTYVGPPGGGGPPGTPIMPSPADSTNSSDNIYTMINPVPPGGSRSNFPMGPGSDGPMGGMGGMEPHHMNGSLGSGDIDGLPKNSPNNISGISNPPGTPRDDGELGGNFLHSFQNDNYSPSMTMSV RPA 1 - Replication protein A 70 kDa DNA-binding subunit(UniProtKB: P27694; NCBI Ref: NM_002945.3) (SEQ ID NO: 368)MVGQLSEGAIAAIMQKGDTNIKPILQVINIRPITTGNSPPRYRLLMSDGLNTLSSFMLATQLNPLVEEEQLSSNCVCQIHRFIVNTLKDGRRVVILMELEVLKSAEAVGVKIGNPVPYNEGLGQPQVAPPAPAASPAASSRPQPQNGSSGMGSTVSKAYGASKTFGKAAGPSLSHTSGGTQSKVVPIASLTPYQSKWTICARVTNKSQIRTWSNSRGEGKLFSLELVDESGE1RATAFNEQVDKFFPLIEVNKVYYFSKGTLKIANKQFTAVKNDYEMTFNNETSVMPCEDDHHLPTVQFDFTGIDDLENKSKDSLVDIIGICKSYEDATKITVRSNNREVAKRNIYLMDTSGKVVTATLWGEDADKFDGSRQPVLAIKGARVSDFGGRSLSVLSSSTIIANPDIPEAYKLRGWFDAEGQALDGVSISDLKSGGVGGSNTNWKTLYEVKSENLGQGDKPDYFSSVATVVYLRKENCMYQACPTQDCNKKVIDQQNGLYRCEKCDTEFPNFKYRMILSVNIADFQENQWVTCFQESAEAILGQNAAYLGELKDKNEQAFEEVFQNANFRSFIFRVRVKVETYNDESRIKATVMDVKPVDYREYGRRL VMSIRRSALMRPA 2 - Replication protein A 32 kDa subunit (UniProtKB: P15927;NCBI Ref: NM_002946) (SEQ ID NO: 369)MWNSGFESYGSSSYGGAGGYTQSPGGFGSPAPSQAEKKSRARAQHIVPCTISQLLSATLVDEVFRIGNVEISQVTIVGIIRHAEKAPTNIVYKIDDMTAAPMDVRQWVDTDDTSSENTVVPPETYVKVAGHLRSFQNKKSLVAFKIMPLEDMNEFTTHILEVINAHMVLSKANSQPSAGRAPISNPGMSEAGNFGGNSFMPANGLTVAQNQVLNLIKACPRPEGLNFQDLKNQLKHMSVSSIKQAVDFLSNEGHIYSTVDDDHFKSTDAERPA 3 - Replication protein A 14 kDa subunit (UniProtKB: P35244;NCBI Ref: NM_002947.4) (SEQ ID NO: 370)MVDMMDLPRSRINAGMLAQFIDKPVCFVGRLEKIHPTGKMFILSDGEGKNGTIELMEPLDEEISGIVEVVGRVTAKATILCTSYVQFKEDSHPFDLGLYNEAVKIIHDFPQFYPLGIVQH DBacterial single-stranded DNA-binding proteins:ssbA - single-stranded DNA-binding protein [Bacillus subtilissubsp. subtilis str. 168] (UniProtKB: P37455; NCBI Ref:)(SEQ ID NO: 371)MLNRVVLVGRLTKDPELRYTPNGAAVATFTLAVNRTFTNQSGEREADFINCVTWRRQAENVANFLKKGSLAGVDGRLQTRNYENQQGQRVFVTEVQAESVQFLEPKNGGGSGSGGYNEGNSGGGQYFGGGQNDNPFGGNQNNQRRNQGNSFNDDPFANDGKPIDISDDDLPFSingle-stranded DNA-binding protein 2 [Streptomyces coelicolorA3(2)] (UniProtKB: Q9X8U3; NCBI Ref: NP_628093.1) (SEQ ID NO: 372)MAGETVITVVGNLVDDPELRFTPSGAAVAKFRVASTPRTFDRQTNEWKDGESLFLTCSVWRQAAENVAESLQRGMRVIVQGRLKQRSYEDREGVKRTVYELDVDEVGASLRSATAKVTKTSGQGRGGQGGYGGGGGGQGGGGWGGGPGGGQQGGGAPADDPWATGGAPAGGQQGGGGQGGGGWGGGSGGGGGYSDEPPFSingle-stranded DNA-binding protein [Streptococcus pneumoniae R6](UniProtKB: P66855; NCBI Ref: NP_358988.1) (SEQ ID NO: 373)MINNVVLVGRMTRDAELRYTPSNVAVATFTLAVNRTFKSQNGEREADFINVVMWRQQAENLANWAKKGSLIGVTGRIQTRSYDNQQGQRVYVTEVVAENFQMLESRSVREGHTGGAYSAPTANYSAPTNSVPDFSRNENPFGATNPLDISDDDLPFViral single-stranded DNA-binding proteins:Single-stranded DNA-binding protein [Human alphaherpesvirus 1](UniProtKB: P04296; NCBI Ref: YP_009137104.1) (SEQ ID NO: 374)METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAPLVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGGRHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGLDPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQLFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAVALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRDGGGKGPAGGFEQRLASVMAGDAALALESIVSMAVFDEPPTDISAWPLFEGQDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHSKPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEHLAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCNLCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLGNYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAMGRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEANHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEGRNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTAPAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSAYQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRNQLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMDAVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLCEQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEEMMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCEDDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDMLSingle-stranded DNA-binding protein from Bacillus virus phi29(UniProtKB: Q38504.1; NCBI Ref: YP_002004532.1) (SEQ ID NO: 375)MENTNIVKATFDTETLEGQIKIFNAQTGGGQSFKNLPDGTIIEANAIAQYKQVSDTYGDAKEETVTTIFAADGSLYSAISKTVAEAASDLIDLVTRHKLETFKVKVVQGTSSKGNVFFSLQLSLSingle stranded DNA binding protein [Burkholderia virus DC1](UniProtKB: I6NRL7; NCBI Ref: YP_006589943.1) (SEQ ID NO: 376)MASVNKVILVGNLGADPETRYLPSGDAISNIRLATTDRYKDKASGEMKESTEWHRVSFFGRLAEIVDEYLRKGAPVYIEGRIRTRKWQDNAGQDRYTTEIVAEKMQMLGDRRDGGERQQRAPQQQQQRTQRNGYADATGRAQPSQRPAAGGGFDEMDDDIPF

Fusion Proteins

Some aspects of the disclosure provide fusion proteins comprising (i) anucleic acid programmable DNA binding protein (napDNAbp), (ii) acytidine deaminase domain and (iii) a Gam protein. In some embodiments,the fusion protein further comprises a uracil glycosylase inhibitor(UGI) domain. In some embodiments, the nucleic acid programmable DNAbinding protein is any of the nucleic acid programmable DNA bindingproteins, or variants thereof, provided herein (e.g., nCas9). In someembodiments, the napDNAbp is a Cas9 domain. Any of the Cas9 domains(e.g., a nuclease active Cas9 protein, a nuclease-inactive dCas9protein, or a Cas9 nickase protein) disclosed herein may be the napDNAbpof any of the fusion proteins, or variants thereof, provided herein. Insome embodiments, the cytidine deaminase domain is any of the cytidinedeaminase domainss, or variants thereof, provided herein. In someembodiments, the cytidine deaminase domain is an APOBEC cytidinedeaminase domain (e.g. rAPOBEC1). In some embodiments, the Gam proteinis any of the Gam proteins, or variants thereof, provided herein. Insome embodiments, the Gam protein is a Gam from bacteriophage Mu. Insome embodiments, the UGI domain is any of the UGI domains, or variantsthereof, provided herein. In some embodiments, the UGI domain comprisesan amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, 98%,or 98%) identical to SEQ ID NO: 362. In some embodiments, the UGI domaincomprises the amino acid sequence of SEQ ID NO: 362.

Some aspects of the disclosure provide fusion proteins comprising anucleic acid programmable DNA binding protein (napDNAbp) a cytidinedeaminase domain and a Gam protein. In some embodiments, any of thefusion proteins provided herein are base editors. In some embodiments,the napDNAbp is a Cas9 domain, a Cpf1 domain, a CasX domain, a CasYdomain, a C2c1 domain, a C2c2 domain, aC2c3 domain, or an Argonautedomain. In some embodiments, the napDNAbp is any napDNAbp providedherein. In some embodiments, the napDNAbp is a Cas9 domain, which may beany of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) providedherein. In some embodiments, any of the Cas9 domains or Cas9 proteins(e.g., dCas9 or nCas9) provided herein may be fused with any of thecytidine deaminases provided herein. In some embodiments, the fusionprotein comprises the structure:

NH₂-[Gam protein]-[cytidine deaminase domain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[napDNAbp]-[cytidine deaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[Gam protein]-COOH; orNH₂-[napDNAbp]-[Gam protein]-[cytidine deaminase domain]-COOH;

In some embodiments, the fusion protein further comprises a UGI domain.In some embodiments, the fusion protein further comprises one or more(e.g., 2, 3, 4, or 5) UGI domains. In some embodiments, the UGI domainis any of the UGI domains, or variants thereof, provided herein. In someembodiments, the fusion protein comprises the structure:

NH₂-[Gam protein]-[cytidine deaminase domain]-[napDNAbp]-[UGIdomain]-COOH;NH₂-[Gam protein]-[napDNAbp]-[cytidine deaminase domain]-[UGIdomain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[napDNAbp]-[UGIdomain]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[Gam protein]-[UGIdomain]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[Gam protein]-[UGIdomain]-COOH;NH₂-[napDNAbp]-[Gam protein]-[cytidine deaminase domain]-[UGIdomain]-COOH;NH₂-[Gam protein]-[cytidine deaminase domain]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[napDNAbp]-[UGI domain]-[cytidine deaminasedomain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[UGI domain]-[Gamprotein]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[UGI domain]-[Gamprotein]-COOH;NH₂-[napDNAbp]-[Gam protein]-[UGI domain]-[cytidine deaminasedomain]-COOH;NH₂-[Gam protein]-[UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[UGI domain]-[napDNAbp]-[cytidine deaminasedomain]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[napDNAbp]-[Gamprotein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[cytidine deaminase domain]-[Gamprotein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[Gam protein]-[cytidine deaminasedomain]-COOH;NH₂-[UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[UGI domain]-[Gam protein]-[napDNAbp]-[cytidine deaminasedomain]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[napDNAbp]-[Gamprotein]-COOH;NH₂-[UGI domain]-[napDNAbp]-[cytidine deaminase domain]-[Gamprotein]-COOH; orNH₂-[UGI domain]-[napDNAbp]-[Gam protein]-[cytidine deaminasedomain]-COOH;

In some embodiments, the fusion protein further comprises a second UGIdomain. In some embodiments, the second UGI domain is any of the UGIdomains, or variants thereof, provided herein. In some embodiments, thesecond UGI domain is the same as the UGI domain. In some embodiments,the second UGI domain is different from the UGI domain. In someembodiments, the fusion protein comprises the structure:

NH₂-[Gam protein]-[cytidine deaminase domain]-[napDNAbp]-[UGIdomain]-[second UGI domain]-COOH;NH₂-[Gam protein]-[napDNAbp]-[cytidine deaminase domain]-[UGIdomain]-[second UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[napDNAbp]-[UGIdomain]-[second UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[Gam protein]-[UGIdomain]-[second UGI domain]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[Gam protein]-[UGIdomain]-[second UGI domain]-COOH;NH₂-[napDNAbp]-[Gam protein]-[cytidine deaminase domain]-[UGIdomain]-[second UGI domain]-COOH;NH₂-[Gam protein]-[cytidine deaminase domain]-[UGIdomain]-[napDNAbp]-[second UGI domain]-COOH;NH₂-[Gam protein]-[napDNAbp]-[UGI domain]-[cytidine deaminasedomain]-[second UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[UGIdomain]-[napDNAbp]-[second UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[UGI domain]-[Gamprotein]-[second UGI domain]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[UGI domain]-[Gamprotein]-[second UGI domain]-COOH;NH₂-[napDNAbp]-[Gam protein]-[UGI domain]-[cytidine deaminasedomain]-[second UGI domain]-COOH;NH₂-[Gam protein]-[UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-[second UGI domain]-COOH;NH₂-[Gam protein]-[UGI domain]-[napDNAbp]-[cytidine deaminasedomain]-[second UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[Gamprotein]-[napDNAbp]-[second UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[napDNAbp]-[Gamprotein]-[second UGI domain]-COOH;NH₂-[napDNAbp]-[UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[second UGI domain]-COOH;NH₂-[napDNAbp]-[UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[second UGI domain]-COOH;NH₂-[UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[napDNAbp]-[second UGI domain]-COOH;NH₂-[UGI domain]-[Gam protein]-[napDNAbp]-[cytidine deaminasedomain]-[second UGI domain]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[napDNAbp]-[second UGI domain]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[napDNAbp]-[Gamprotein]-[second UGI domain]-COOH;NH₂-[UGI domain]-[napDNAbp]-[cytidine deaminase domain]-[Gamprotein]-[second UGI domain]-COOH;NH₂-[UGI domain]-[napDNAbp]-[Gam protein]-[cytidine deaminasedomain]-[second UGI domain]-COOH;NH₂-[second UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[napDNAbp]-[UGI domain]-COOH;NH₂-[second UGI domain]-[Gam protein]-[napDNAbp]-[cytidine deaminasedomain]-[UGI domain]-COOH;NH₂-[second UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[napDNAbp]-[UGI domain]-COOH;NH₂-[second UGI domain]-[cytidine deaminase domain]-[napDNAbp]-[Gamprotein]-[UGI domain]-COOH;NH₂-[second UGI domain]-[napDNAbp]-[cytidine deaminase domain]-[Gamprotein]-[UGI domain]-COOH;NH₂-[second UGI domain]-[napDNAbp]-[Gam protein]-[cytidine deaminasedomain]-[UGI domain]-COOH;NH₂-[second UGI domain]-[Gam protein]-[cytidine deaminase domain]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[second UGI domain]-[Gam protein]-[napDNAbp]-[UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[second UGI domain]-[cytidine deaminase domain]-[Gam protein]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[second UGI domain]-[cytidine deaminase domain]-[napDNAbp]-[UGIdomain]-[Gam protein]-COOH;NH₂-[second UGI domain]-[napDNAbp]-[cytidine deaminase domain]-[UGIdomain]-[Gam protein]-COOH;NH₂-[second UGI domain]-[napDNAbp]-[Gam protein]-[UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[second UGI domain]-[Gam protein]-[UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[second UGI domain]-[Gam protein]-[UGI domain]-[napDNAbp]-[cytidinedeaminase domain]-COOH;NH₂-[second UGI domain]-[cytidine deaminase domain]-[UGI domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[second UGI domain]-[cytidine deaminase domain]-[UGIdomain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[second UGI domain]-[napDNAbp]-[UGI domain]-[cytidine deaminasedomain]-[Gam protein]-COOH;NH₂-[second UGI domain]-[napDNAbp]-[UGI domain]-[Gam protein]-[cytidinedeaminase domain]-COOH;NH₂-[second UGI domain]-[UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[second UGI domain]-[UGI domain]-[Gam protein]-[napDNAbp]-[cytidinedeaminase domain]-COOH;NH₂-[second UGI domain]-[UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[second UGI domain]-[UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[second UGI domain]-[UGI domain]-[napDNAbp]-[cytidine deaminasedomain]-[Gam protein]-COOH;NH₂-[second UGI domain]-[UGI domain]-[napDNAbp]-[Gam protein]-[cytidinedeaminase domain]-COOH;NH₂-[Gam protein]-[second UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-[UGI domain]-COOH;NH₂-[Gam protein]-[second UGI domain]-[napDNAbp]-[cytidine deaminasedomain]-[UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[Gamprotein]-[napDNAbp]-[UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[napDNAbp]-[Gamprotein]-[UGI domain]-COOH;NH₂-[napDNAbp]-[second UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[UGI domain]-COOH;NH₂-[napDNAbp]-[second UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[UGI domain]-COOH;NH₂-[Gam protein]-[second UGI domain]-[cytidine deaminase domain]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[second UGI domain]-[napDNAbp]-[UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[Gam protein]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[napDNAbp]-[UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[second UGI domain]-[cytidine deaminase domain]-[UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[second UGI domain]-[Gam protein]-[UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[Gam protein]-[second UGI domain]-[UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[second UGI domain]-[UGI domain]-[napDNAbp]-[cytidinedeaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[UGI domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[UGIdomain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[second UGI domain]-[cytidine deaminasedomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[second UGI domain]-[Gam protein]-[cytidinedeaminase domain]-COOH;NH₂-[UGI domain]-[second UGI domain]-[Gam protein]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[UGI domain]-[second UGI domain]-[Gam protein]-[napDNAbp]-[cytidinedeaminase domain]-COOH;NH₂-[UGI domain]-[second UGI domain]-[cytidine deaminase domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[UGI domain]-[second UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[UGI domain]-[second UGI domain]-[napDNAbp]-[cytidine deaminasedomain]-[Gam protein]-COOH;NH₂-[UGI domain]-[second UGI domain]-[napDNAbp]-[Gam protein]-[cytidinedeaminase domain]-COOH;NH₂-[Gam protein]-[cytidine deaminase domain]-[second UGIdomain]-[napDNAbp]-[UGI domain]-COOH;NH₂-[Gam protein]-[napDNAbp]-[second UGI domain]-[cytidine deaminasedomain]-[UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[Gamprotein]-[napDNAbp]-[UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[second UGI domain]-[napDNAbp]-[Gamprotein]-[UGI domain]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[second UGI domain]-[Gamprotein]-[UGI domain]-COOH;NH₂-[napDNAbp]-[Gam protein]-[second UGI domain]-[cytidine deaminasedomain]-[UGI domain]-COOH;NH₂-[Gam protein]-[cytidine deaminase domain]-[second UGI domain]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[napDNAbp]-[second UGI domain]-[UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[second UGI domain]-[UGIdomain]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[second UGI domain]-[UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[second UGI domain]-[UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[Gam protein]-[second UGI domain]-[UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[Gam protein]-[UGI domain]-[second UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[UGI domain]-[second UGI domain]-[napDNAbp]-[cytidinedeaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[second UGI domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[second UGIdomain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[second UGI domain]-[cytidine deaminasedomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[second UGI domain]-[Gam protein]-[cytidinedeaminase domain]-COOH;NH₂-[UGI domain]-[Gam protein]-[second UGI domain]-[cytidine deaminasedomain]-[napDNAbp]-COOH;NH₂-[UGI domain]-[Gam protein]-[second UGI domain]-[napDNAbp]-[cytidinedeaminase domain]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[second UGI domain]-[Gamprotein]-[napDNAbp]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[second UGIdomain]-[napDNAbp]-[Gam protein]-COOH;NH₂-[UGI domain]-[napDNAbp]-[second UGI domain]-[cytidine deaminasedomain]-[Gam protein]-COOH;NH₂-[UGI domain]-[napDNAbp]-[second UGI domain]-[Gam protein]-[cytidinedeaminase domain]-COOH;NH₂-[Gam protein]-[cytidine deaminase domain]-[napDNAbp]-[second UGIdomain]-[UGI domain]-COOH;NH₂-[Gam protein]-[napDNAbp]-[cytidine deaminase domain]-[second UGIdomain]-[UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[napDNAbp]-[second UGIdomain]-[UGI domain]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[Gam protein]-[second UGIdomain]-[UGI domain]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[Gam protein]-[second UGIdomain]-[UGI domain]-COOH;NH₂-[napDNAbp]-[Gam protein]-[cytidine deaminase domain]-[second UGIdomain]-[UGI domain]-COOH;NH₂-[Gam protein]-[cytidine deaminase domain]-[UGI domain]-[second UGIdomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[napDNAbp]-[UGI domain]-[second UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[Gam protein]-[UGI domain]-[second UGIdomain]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[napDNAbp]-[UGI domain]-[second UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[cytidine deaminase domain]-[UGI domain]-[second UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[Gam protein]-[UGI domain]-[second UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[Gam protein]-[UGI domain]-[cytidine deaminase domain]-[second UGIdomain]-[napDNAbp]-COOH;NH₂-[Gam protein]-[UGI domain]-[napDNAbp]-[second UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[Gam protein]-[second UGIdomain]-[napDNAbp]-COOH;NH₂-[cytidine deaminase domain]-[UGI domain]-[napDNAbp]-[second UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[cytidine deaminase domain]-[second UGIdomain]-[Gam protein]-COOH;NH₂-[napDNAbp]-[UGI domain]-[Gam protein]-[second UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[UGI domain]-[Gam protein]-[cytidine deaminase domain]-[second UGIdomain]-[napDNAbp]-COOH;NH₂-[UGI domain]-[Gam protein]-[napDNAbp]-[second UGI domain]-[cytidinedeaminase domain]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[Gam protein]-[second UGIdomain]-[napDNAbp]-COOH;NH₂-[UGI domain]-[cytidine deaminase domain]-[napDNAbp]-[second UGIdomain]-[Gam protein]-COOH;NH₂-[UGI domain]-[napDNAbp]-[cytidine deaminase domain]-[second UGIdomain]-[Gam protein]-COOH; orNH₂-[UGI domain]-[napDNAbp]-[Gam protein]-[second UGI domain]-[cytidinedeaminase domain]-COOH;

In some embodiments, the fusion proteins provided herein, for exampleany of the fusion proteins comprising the above structures, do notinclude a linker sequence. In some embodiments, a linker is presentbetween any of the proteins or domains provided herein. In someembodiments, the “-” used in the structures above indicates the presenceof an optional linker. In some embodiments, the “-” may be any of thelinkers provided herein. In some embodiments, the Gam protein and thecytidine deaminase domain are fused via any of the linkers providedherein. In some embodiments, the Gam protein and the napDNAbp are fusedvia any of the linkers provided herein. In some embodiments, the Gamprotein and the UGI domain are fused via any of the linkers providedherein. In some embodiments, the Gam protein and the second UGI domainare fused via any of the linkers provided herein.

In some embodiments, the cytidine deaminase domain and the Gam proteinare fused via any of the linkers provided herein. In some embodiments,the cytidine deaminase domain and the napDNAbp are fused via any of thelinkers provided herein. In some embodiments, the cytidine deaminasedomain and the UGI domain are fused via any of the linkers providedherein. In some embodiments, the cytidine deaminase domain and thesecond UGI domain are fused via any of the linkers provided herein. Insome embodiments, the napDNAbp and the Gam protein are fused via any ofthe linkers provided herein. In some embodiments, the napDNAbp and thecytidine deaminase domain are fused via any of the linkers providedherein. In some embodiments, the napDNAbp and the UGI domain are fusedvia any of the linkers provided herein. In some embodiments, thenapDNAbp and the second UGI domain are fused via any of the linkersprovided herein. In some embodiments, the UGI domain and the Gam proteinare fused via any of the linkers provided herein. In some embodiments,the UGI domain and the cytidine deaminase domain are fused via any ofthe linkers provided herein. In some embodiments, the UGI domain and thenapDNAbp domain are fused via any of the linkers provided herein. Insome embodiments, the UGI domain and the second UGI domain are fused viaany of the linkers provided herein. In some embodiments, the second UGIdomain and the Gam protein are fused via any of the linkers providedherein. In some embodiments, the second UGI domain and the cytidinedeaminase domain are fused via any of the linkers provided herein. Insome embodiments, the second UGI domain and the napDNAbp domain arefused via any of the linkers provided herein. In some embodiments, thesecond UGI domain and the UGI domain are fused via any of the linkersprovided herein.

For example, in some embodiments the domains and/or proteins describedabove are fused via any of the linkers provided below in the sectionentitled “Linkers”. In some embodiments, any of the domains and/orproteins provided herein are fused via a linker that comprises between 1and and 200 amino acids. In some embodiments, any of the domains and/orproteins provided herein are fused via a linker that comprises orconsists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, or more amino acids in length. In some embodiments,any of the domains and/or proteins provided herein are fused via alinker that comprises from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20,5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40to 150, 40 to 200, 50 to 60 50 to 80, 50 to 100, 50 to 150, 50 to 200,60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. Insome embodiments, any of the domains and/or proteins provided herein arefused via a linker that comprises 4, 9, 10, 16, or 32, amino acids inlength. In some embodiments, any of the domains and/or proteins providedherein are fused via a linker that comprises the amino acid sequence ofSGSETPGTSESATPES (SEQ ID NO: 377), SGGS (SEQ ID NO: 378),SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 379),SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 380), orGGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 381). In someembodiments, any of the domains and/or proteins provided herein arefused via a linker comprising the amino acid sequence SGSETPGTSESATPES(SEQ ID NO: 377), which may also be referred to as the XTEN linker. Insome embodiments, the linker is 24 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 382). In some embodiments, thelinker is 40 amino acids in length. In some embodiments, the linkercomprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO:383). In someembodiments, the linker is 64 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGG SSGGS (SEQID NO: 384). In some embodiments, the linker is 92 amino acids inlength. In some embodiments, the linker comprises the amino acidsequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 385). In some embodiments,the linker is 10 amino acids in length. In some embodiments, the linkercomprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 386)

In some embodiments, the Gam protein and the cytidine deaminase domainof any of the fusion proteins provided herein are fused via a linkercomprising from 10-20 amino acids in length. In some embodiments, theGam protein and the cytidine deaminase domain of any of the fusionproteins provided herein are fused via a linker comprising 16 aminoacids in length. In some embodiments, the Gam protein and the cytidinedeaminase domain of any of the fusion proteins provided herein are fusedvia a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ IDNO: 377).

In some embodiments, the cytidine deaminase domain and the napDNAbp ofany of the fusion proteins provided herein are fused via a linkercomprising from 10-20 amino acids in length, or from 25-40 amino acidsin length. In some embodiments, the cytidine deaminase domain and thenapDNAbp of any of the fusion proteins provided herein are fused via alinker comprising 16 or 32 amino acids in length. In some embodiments,the cytidine deaminase domain and the napDNAbp of any of the fusionproteins provided herein are fused via a linker comprising the aminoacid sequence SGSETPGTSESATPES (SEQ ID NO: 377) orSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 380).

In some embodiments, the napDNAbp and the UGI domain of any of thefusion proteins provided herein are fused via a linker comprising from1-8 amino acids in length, or from 4-15 amino acids in length. In someembodiments, the napDNAbp and the UGI domain of any of the fusionproteins provided herein are fused via a linker comprising 4, 9 or 10amino acids in length. In some embodiments, the napDNAbp and the UGIdomain of any of the fusion proteins provided herein are fused via alinker comprising the amino acid sequence SGGSGGSGGS (SEQ ID NO: 386) orSGGS (SEQ ID NO: 378).

In some embodiments, the UGI domain and the second UGI domain of any ofthe fusion proteins provided herein are fused via a linker comprisingfrom 2-15 amino acids in length. In some embodiments, the UGI domain andthe second UGI domain of any of the fusion proteins provided herein arefused via a linker comprising 9 or 10 amino acids in length. In someembodiments, the UGI domain and the second UGI domain of any of thefusion proteins provided herein are fused via a linker comprising theamino acid sequence SGGSGGSGGS (SEQ ID NO: 386).

Linkers

In certain embodiments, linkers may be used to link any of the proteinsor protein domains described herein. The linker may be as simple as acovalent bond, or it may be a polymeric linker many atoms in length. Incertain embodiments, the linker is a polypeptide or based on aminoacids. In other embodiments, the linker is not peptide-like. In certainembodiments, the linker is a covalent bond (e.g., a carbon-carbon bond,disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments,the linker is a carbon-nitrogen bond of an amide linkage. In certainembodiments, the linker is a cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic or heteroaliphaticlinker. In certain embodiments, the linker is polymeric (e.g.,polyethylene, polyethylene glycol, polyamide, polyester, etc.). Incertain embodiments, the linker comprises a monomer, dimer, or polymerof aminoalkanoic acid. In certain embodiments, the linker comprises anaminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine,3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). Incertain embodiments, the linker comprises a monomer, dimer, or polymerof aminohexanoic acid (Ahx). In certain embodiments, the linker is basedon a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In otherembodiments, the linker comprises a polyethylene glycol moiety (PEG). Inother embodiments, the linker comprises amino acids. In certainembodiments, the linker comprises a peptide. In certain embodiments, thelinker comprises an aryl or heteroaryl moiety. In certain embodiments,the linker is based on a phenyl ring. The linker may includefunctionalized moieties to facilitate attachment of a nucleophile (e.g.,thiol, amino) from the peptide to the linker. Any electrophile may beused as part of the linker. Exemplary electrophiles include, but are notlimited to, activated esters, activated amides, Michael acceptors, alkylhalides, aryl halides, acyl halides, and isothiocyanates.

In some embodiments, the linker is an amino acid or a plurality of aminoacids (e.g., a peptide or protein). In some embodiments, the linker is abond (e.g., a covalent bond), an organic molecule, group, polymer, orchemical moiety. In some embodiments, the linker is 2-100 amino acids inlength, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40,40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer orshorter linkers are also contemplated. In some embodiments, a linkercomprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 377),which may also be referred to as the XTEN linker. In some embodiments, alinker comprises the amino acid sequence SGGS (SEQ ID NO: 378). In someembodiments, a linker comprises (SGGS)_(n) (SEQ ID NO: 387), (GGGS)_(n)(SEQ ID NO: 388), (GGGGS)_(n) (SEQ ID NO: 389), (G)_(n), (EAAAK)_(n)(SEQ ID NO: 390), (GGS). (SEQ ID NO:391), SGSETPGTSESATPES (SEQ ID NO:377), or (XP)_(n) motif, or a combination of any of these, wherein n isindependently an integer between 1 and 30, and wherein X is any aminoacid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15. In some embodiments, a linker comprises SGSETPGTSESATPES(SEQ ID NO: 377), and SGGS (SEQ ID NO: 378). In some embodiments, alinker comprises SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 379). In someembodiments, a linker comprises SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ IDNO: 380). In some embodiments, a linker comprisesGGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 381). In someembodiments, the linker is 24 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 382). In some embodiments, thelinker is 40 amino acids in length. In some embodiments, the linkercomprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 383). In someembodiments, the linker is 64 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGG SSGGS (SEQID NO: 384). In some embodiments, the linker is 92 amino acids inlength. In some embodiments, the linker comprises the amino acidsequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 385). It should beappreciated that any of the linkers provided herein may be used to linka Gam protein and a cytidine deaminase domain, a cytidine deaminasedomain and a napDNAbp, a napDNAbp and a UGI domain, and/or a UGI domainand a second UGI domain in any of the fusion proteins provided herein.

NLS

Some aspects of the disclosure provide fusion proteins (e.g., any of thefusion proteins provided herein) that comprise one or more NLSs. Theterm “nuclear localization sequence” or “NLS” refers to an amino acidsequence that promotes import of a protein into the cell nucleus, forexample, by nuclear transport. Nuclear localization sequences are knownin the art and would be apparent to the skilled artisan. For example,NLS sequences are described in Plank et al., international PCTapplication, PCT/EP2000/011690, filed Nov. 23, 2000, publishedasWO/2001/038547 on May 31, 2001, the contents of which are incorporatedherein by reference for their disclosure of exemplary nuclearlocalization sequences. In some embodiments, the NLS is a bipartitenuclear localization sequence (BPNLS), e.g., KRTADGSEFEPKKKRKV (SEQ IDNO: 405). In some embodiments, a NLS comprises the amino acid sequencePKKKRKV (SEQ ID NO: 392), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO:393), or KRTADGSEFEPKKKRKV (SEQ ID NO: 405).

In some embodiments, fusion proteins provided herein further comprise anuclear localization sequence (NLS). In some embodiments, the NLS isfused to the N-terminus of the fusion protein. In some embodiments, theNLS is fused to the C-terminus of the fusion protein. In someembodiments, the NLS is fused to the N-terminus of the UGI domain. Insome embodiments, the NLS is fused to the C-terminus of the UGI domain.In some embodiments, the NLS is fused to the N-terminus of the napDNAbp.In some embodiments, the NLS is fused to the C-terminus of the napDNAbp.In some embodiments, the NLS is fused to the N-terminus of the cytidinedeaminase domain. In some embodiments, the NLS is fused to theC-terminus of the cytidine deaminase domain. In some embodiments, theNLS is fused to the N-terminus of the Gam protein. In some embodiments,the NLS is fused to the C-terminus of the Gam protein. In someembodiments, the NLS is fused to the N-terminus of the second UGIdomain. In some embodiments, the NLS is fused to the C-terminus of thesecond UGI domain. In some embodiments, the NLS is fused to the fusionprotein via one or more linkers. In some embodiments, the NLS is fusedto the fusion protein without a linker. In some embodiments, the NLScomprises an amino acid sequence of any one of the NLS sequencesprovided or referenced herein. In some embodiments, the NLS comprises anamino acid sequence PKKKRKV (SEQ ID NO: 392),MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 393), or KRTADGSEFEPKKKRKV(SEQ ID NO: 405).

Some aspects of the disclosure provide fusion proteins that are capableof editing a base within a nucleic acid molecule (e.g., DNA or RNA). Insome embodiments, any of the fusion proteins provided herein are baseeditors. Exemplary base editors are provided below. For Example, theamino acid sequences of BE3, BE4, BE3-Gam and BE4-Gam, which aredescribed in the Examples, are provided below. In some embodiments, thefusion protein comprises an amino acid sequence that is at least atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% of the amino acid sequence ofany of the fusion proteins provided herein. In some embodiments, afusion protein comprises an amino acid sequence that is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to the amino acid sequence ofBE3-Gam or BE4-Gam, provided below. In some embodiments, the fusionprotein comprises the amino acid sequence of BE3-Gam or BE4-Gam,provided below.

Exemplary Base Editors:

Amino acid sequences of exemplary base editors (e.g., for BE3-Gam, BE4,and BE4-Gam) are provided below. Domains of the below base editors areidentified, for the purposes of clarity, using text formatting. Gam isindicated in bold text, linkers are indicated by underlining, thecytidine deaminase domain (e.g., APOBEDC1) is indicated in italics, thenapDNAbp (e.g., nCas9) is indicated in unformatted text, the UGI domainis indicated in bold italics, and the NLS is indicated in doubleunderlining. It should be appreciated that amino acid sequences providedherein, for example base editors, cytidine deaminases, napDNAbps, Gamproteins, and UGI domains need not include an N-terminal methionine (M)residue. Accordingly, the disclosure contemplates any of the proteinsprovided herein absent an N-terminal M residue.

BE3: (SEQ ID NO: 397)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK SGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGS

SGGS PKKKRKV BE3-Gam: (SEQ ID NO: 394)MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIPFEQEAGI SGSETPGTSESATPES SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCHLGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK SGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGS

SGGS PKKKRKV BE4: (SEQ ID NO: 395)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK SGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGS

SGGSGGSGGS

SGGS PKKKRK BE4-Gam: (SEQ ID NO: 396)MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIPFEQEAGI SGSETPGTSESATPES SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCHLGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKS GGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDATLLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGS

SGGSGG SGGS

SGGS PKKKRK

The description of exemplary embodiments of the above disclosure isprovided for illustration purposes only and not meant to be limiting.Additional fusion proteins and methods of using the same, e.g.,variations of the exemplary systems described in detail above, are alsoembraced by this disclosure.

EXAMPLES Example 1 UNG Activity is Required for Byproduct Formation

It was hypothesized that undesired base editing byproducts arise duringbase excision repair due to the formation and error-prone resolution ofabasic sites within the uracil-containing DNA strand. This hypothesispredicts that the product purity of base editing in cells lacking uracilNglycosylase (UNG) should be greatly improved. To test this prediction,HAP1 cells (a haploid human cell line) and HAP1 UNG— cells werenucleofected with plasmids encoding BE3 and sgRNAs targeting the EMX1,FANCF, HEK2, HEK3, HEK4, or RNF2 loci (see FIG. 1B for targetsequences). Three days post-nucleofection, genomic DNA was extracted andthe target loci were amplified by PCR and analyzed by high-throughputDNA sequencing (HTS). Base editing product purity is defined as thepercent of edited sequencing reads (reads in which the target C has beenconverted to A, G, or T) in which the target C is edited to a T. Thebase editing product purity of BE3-treated HAP1 cells averaged 68±6%(mean±s.d. for n=3 biological replicates) across 12 target Cs in the sixloci. Remarkably, in HAP1 UNG— cells, all 12 target Cs tested were baseedited with product purities >98% (FIG. 1A). In addition, indelfrequencies at all six tested loci decreased 7- to 100-fold upon UNGknockout (FIG. 1C). These data strongly implicate UNG activity asnecessary for undesired product formation during base editing,consistent with a model in which abasic site formation and subsequentbase excision repair with error-prone polymerases leads to randomizationof the target nucleotide and occasional strand breaks that result inindels.

Fusion with Gam Further Reduces Inde1 Frequencies and Improves ProductPurity

For some genome editing applications, the formation of indels confoundsresearch or poses safety risks. It was therefore sought to furtherdecrease indel frequencies that arise from base editing. It washypothesized that the majority of base editing-induced indels occur as aresult of DNA-(apurinic or apyrimidinic site) lyase (AP lyase), a BERenzyme that converts abasic sites into ssDNA nicks (25). Since baseeditors nick the strand opposite the U, cleavage of the glycosidic bondby UNG followed by processing of the resulting AP site by AP lyase wouldresult in a DSB, which promotes indel formation. This model isconsistent with the observation of greatly reduced indel frequencies inUNG knockout cells (FIG. 1C). The Gam protein of Mu bacteriophage bindsto the ends of DSB s and protects them from degradation (27), and hasbeen repurposed to image DSBs in live mammalian cells (28). It wasreasoned that using Gam to bind the free ends of DSB may reduce indelformation during the process of base editing. Thus, the 174-residue Gamprotein was fused to the N-terminus of base editors BE3 and BE4 via the16-amino acid XTEN linker to generate BE3-Gam and BE4-Gam, respectively.

BE3-Gam decreased indel frequencies relative to BE3 at all six genomicloci tested by an average of 1.7±0.3-fold (FIG. 2C). C-to-T editingefficiencies for BE3-Gam were similar to or higher than those of BE3(FIG. 2B). In addition, BE3-Gam also exhibited increased product purityrelative to BE3 at all genomic loci tested, with an average decrease innon-T product formation of 1.5±0.1-fold (FIG. 2D).

BE4-Gam exhibited greatly decreased indel frequencies relative to BE4 atall genomic loci tested, with an average decrease of 3.3±0.8-fold (FIG.2C). In general, indel frequency following BE4-Gam treatment is below0.5%. We observed decreases in C-to-T editing efficiencies for BE4-Gamrelative to BE4 at some loci, averaging 2.2±0.2-fold (FIG. 2B), perhapsdue to the large size (230 kDa) or presence of four linkers withinBE4-Gam. Nonetheless, BE4-Gam offers overall editing:indel ratios thatincrease an average of 1.5±0.3-fold across all six sites relative to BE4(FIG. 3). Product purities of BE4-Gam are similar or improved comparedwith BE4 (FIG. 2D).

For base editing applications in which minimizing indel production iscritical, BE4-Gam or BE3-Gam may be preferred (FIG. 2E). BE4-Gam offersthe lowest indel frequency and highest product purity among the baseeditors tested (FIG. 2E), albeit with reduced editing efficiency. C-to-Tediting efficiency:indel ratios increase as BE3<BE3-Gam <BE4<BE4-Gamacross all six genomic loci (FIG. 3). It was speculated that Gam may beinducing the death of DSB-containing cells, consistent with previousfindings (28), thereby removing indels from the population of treated,surviving cells. FIG. 2E suggests appropriate base editor(s) to use whenbalancing high editing efficiency, high product purity, and low indelfrequency.

Collectively, these developments advance the state-of-the-art inprogrammable C:G to T:A base pair conversion, and thereby increase theutility and applicability of base editing. Findings also suggest thatGam has the potential to be repurposed to minimize indel formation inother genome editing applications. Finally, relationships among uracilincorporation, UNG activity, and cellular DNA repair outcomesilluminated in this study may guide future efforts to understand ormanipulate eukaryotic DNA repair.

Cloning of Plasmids

All plasmids in this study were generated by USER cloning using PhusionU Hot

Start polymerase (Thermo Fisher). Deaminase and SSB genes weresynthesized as gBlocks Gene Fragments (Integrated DNA Technologies), andTarget-AID was obtained from Addgene (plasmid #79620). Protein sequencesare listed in the Supplementary Notes.

Cell Culture

HEK293T (ATCC CRL-3216) cells were maintained in Dulbecco's ModifiedEagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v)fetal bovine serum (FBS), at 37° C. with 5% CO2. HAP1 (Horizon DiscoveryC631) and HAP1 UNG-(Horizon Discovery HZGHC001531c012) were maintainedin Iscove's Modified Dulbecco's Medium plus GlutaMax (ThermoFisherScientific) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37°C. with 5% CO2.

Transfections

HEK293T cells were seeded on 48-well collagen-coated BioCoat plates(Corning) and transfected at approximately 75% confluency. Briefly, 750ng of BE and 250 ng of sgRNA expression plasmids were transfected using1.5 μL of Lipofectamine 2000 (ThermoFisher Scientific) per wellaccording to the manufacturer's protocol. HAP1 and HAP1 UNG— cells werenucleofected using the SE Cell Line 4DNucleofector™ X Kit S (Lonza)according to the manufacturer's protocol. Briefly, 4×10⁵ cells werenucleofected with 300 ng of BE and 100 ng of sgRNA expression plasmidsusing the 4DNucleofector™ program DZ-113.

High-Throughput DNA Sequencing of Genomic DNA Samples

Transfected cells were harvested after 3 d and the genomic DNA wasisolated by incubating cells in lysis buffer (10 mM Tris-HCl pH 8.0,0.05% SDS, 25 μg/mL proteinase K) at 37° C. for 1 hr followed by 80° C.for 30 min. Genomic regions of interest were amplified by PCR withflanking HTS primer pairs as previously described 6,1. PCR amplificationwas carried out with Phusion high-fidelity DNA polymerase (ThermoFisher)according to the manufacturer's instructions and as previouslydescribed. Purified DNA was amplified by PCR with primers containingsequencing adaptors. The products were gel-purified and quantified usingthe QuantiT™ PicoGreen dsDNA Assay Kit (ThermoFisher) and KAPA LibraryQuantification Kit-Illumina (KAPA Biosystems). Samples were sequenced onan Illumina MiSeq as previously described.

Data analysis

Sequencing reads were automatically demultiplexed using MiSeq Reporter(Illumina), and individual FASTQ files were analyzed with a customMatlab script as previously described (1). Each read was pairwisealigned to the appropriate reference sequence using the Smith-Watermanalgorithm. Base calls with a Q-score below 31 were replaced with Ns andwere thus excluded in calculating nucleotide frequencies. This treatmentyields an expected MiSeq basecalling error rate of approximately 1 in1,000. Aligned sequences in which the read and reference sequencecontained no gaps were stored in an alignment table from which basefrequencies could be tabulated for each locus.

Indel frequencies were quantified with the previously described Matlabscript 5,6,1. Briefly, sequencing reads were scanned for exact matchesto two 10-bp sequences that flank both sides of a window in which indelsmight occur. If no exact matches were located, the read was excludedfrom analysis. If the length of this indel window exactly matched thereference sequence the read was classified as not containing an indel.If the indel window was two or more bases longer or shorter than thereference sequence, then the sequencing read was classified as aninsertion or deletion, respectively.

In order to evaluate interdependency (linkage disequilibrium) betweenthe base editing outcomes at the multiple target cytidines within anediting window, target site sequences from BE treated cells wereanalyzed by a custom Python script (Supplementary Note 1). Briefly,sequencing reads were scanned for exact matches to two 7-bp sequencesthat flank each side of the protospacer. If the intervening region wasnot exactly 20-bp, then it was excluded further analysis. Theprotospacer sequences were further filtered into four groups based uponthe identity of the nucleotide at the position with the most non-Tediting outcomes (the primary target C). For each of these four groupsas well as the entire pool, we tallied the nucleotide abundance at eachof the 20 positions within the protospacer.

napDNAbp Complexes with Guide RNAs

Some aspects of this disclosure provide complexes comprising any of thefusion proteins provided herein, and a nucleic acid (e.g., DNA or RNA)bound to the napDNAbp of any of the fusion proteins provided herein.Some aspects of this disclosure provide complexes comprising any of thefusion proteins provided herein, and a guide RNA bound to a Cas9 domain(e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) of fusionprotein.

In some embodiments, the guide RNA is from 15-100 nucleotides long andcomprises a sequence of at least 10 contiguous nucleotides that iscomplementary to a target sequence. In some embodiments, the guide RNAis 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 nucleotides long. In some embodiments, the guide RNA comprises asequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotidesthat is complementary to a target sequence. In some embodiments, thetarget sequence is a DNA sequence. In some embodiments, the targetsequence is a sequence in the genome of a mammal. In some embodiments,the target sequence is a sequence in the genome of a human. In someembodiments, the 3′ end of the target sequence is immediately adjacentto a canonical PAM sequence (NGG). In some embodiments, the guide RNA iscomplementary to a sequence associated with a disease or disorder.

Methods of Using Fusion Proteins

Some aspects of the disclosure provide methods for using the fusionproteins (e.g., base editors) provided herein. Some aspects of thisdisclosure provide methods of using the fusion proteins, or complexesprovided herein. For example, some aspects of this disclosure providemethods comprising contacting a DNA molecule (a) with any of the fusionproteins provided herein, and with at least one guide RNA, wherein theguide RNA is about 15-100 nucleotides long and comprises a sequence ofat least 10 contiguous nucleotides that is complementary to a targetsequence; or (b) with a Cas9 protein, a Cas9 fusion protein, or a Cas9protein or fusion protein complex with at least one gRNA as providedherein. In some embodiments, the 3′ end of the target sequence is notimmediately adjacent to a canonical PAM sequence (NGG). In someembodiments, the 3′ end of the target sequence is immediately adjacentto an AGC, GAG, TTT, GTG, or CAA sequence.

In some embodiments, the target DNA sequence comprises a sequenceassociated with a disease or disorder. In some embodiments, the targetDNA sequence comprises a point mutation associated with a disease ordisorder. In some embodiments, the activity of the fusion protein, orthe complex results in a correction of the point mutation. In someembodiments, the target DNA sequence comprises a T→C point mutationassociated with a disease or disorder, and wherein the deamination ofthe mutant C base results in a sequence that is not associated with adisease or disorder. In some embodiments, the target DNA sequenceencodes a protein and wherein the point mutation is in a codon andresults in a change in the amino acid encoded by the mutant codon ascompared to the wild-type codon. In some embodiments, the deamination ofthe mutant C results in a change of the amino acid encoded by the mutantcodon. In some embodiments, the deamination of the mutant C results inthe codon encoding the wild-type amino acid. In some embodiments, thecontacting is in vivo in a subject. In some embodiments, the subject hasor has been diagnosed with a disease or disorder. In some embodiments,the disease or disorder is cystic fibrosis, phenylketonuria,epidermolytic hyperkeratosis (EHK), Charcot-Marie-Toot disease type 4J,neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital,hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditarylymphedema, familial Alzheimer's disease, HIV, Prion disease, chronicinfantile neurologic cutaneous articular syndrome (CINCA),desmin-related myopathy (DRM), a neoplastic disease associated with amutant PI3KCA protein, a mutant CTNNB1 protein, a mutant HRAS protein,or a mutant p53 protein.

Some embodiments provide methods for using the base editing fusionproteins provided herein. In some embodiments, the fusion protein isused to introduce a point mutation into a nucleic acid by deaminating atarget nucleobase, e.g., a C residue. In some embodiments, thedeamination of the target nucleobase results in the correction of agenetic defect, e.g., in the correction of a point mutation that leadsto a loss of function in a gene product. In some embodiments, thegenetic defect is associated with a disease or disorder, e.g., alysosomal storage disorder or a metabolic disease, such as, for example,type I diabetes. In some embodiments, the methods provided herein areused to introduce a deactivating point mutation into a gene or allelethat encodes a gene product that is associated with a disease ordisorder. For example, in some embodiments, methods are provided hereinthat employ a Cas9 DNA editing fusion protein to introduce adeactivating point mutation into an oncogene (e.g., in the treatment ofa proliferative disease). A deactivating mutation may, in someembodiments, generate a premature stop codon in a coding sequence, whichresults in the expression of a truncated gene product, e.g., a truncatedprotein lacking the function of the full-length protein.

In some embodiments, the purpose of the methods provide herein is torestore the function of a dysfunctional gene via genome editing. Thefusion proteins provided herein can be validated for gene editing-basedhuman therapeutics in vitro, e.g., by correcting a disease-associatedmutation in human cell culture. It will be understood by the skilledartisan that the fusion proteins provided herein, e.g., the fusionproteins comprising a Cas9 domain and a nucleic acid deaminase domaincan be used to correct any single point T to C or A to G mutation. Inthe first case, deamination of the mutant C back to U corrects themutation, and in the latter case, deamination of the C that isbase-paired with the mutant G, followed by a round of replication,corrects the mutation.

An exemplary disease-relevant mutation that can be corrected by theprovided fusion proteins in vitro or in vivo is the H1047R (A3140G)polymorphism in the PI3KCA protein. The phosphoinositide-3-kinase,catalytic alpha subunit (PI3KCA) protein acts to phosphorylate the 3-OHgroup of the inositol ring of phosphatidylinositol. The PI3KCA gene hasbeen found to be mutated in many different carcinomas, and thus it isconsidered to be a potent oncogene. In fact, the A3140G mutation ispresent in several NCI-60 cancer cell lines, such as, for example, theHCT116, SKOV3, and T47D cell lines, which are readily available from theAmerican Type Culture Collection (ATCC).³⁸

In some embodiments, a cell carrying a mutation to be corrected, e.g., acell carrying a point mutation, e.g., an A3140G point mutation in exon20 of the PI3KCA gene, resulting in a H1047R substitution in the PI3KCAprotein, is contacted with an expression construct encoding a baseediting fusion protein and an appropriately designed sgRNA targeting thefusion protein to the respective mutation site in the encoding PI3KCAgene. Control experiments can be performed where the sgRNAs are designedto target the fusion enzymes to non-C residues that are within thePI3KCA gene. Genomic DNA of the treated cells can be extracted, and therelevant sequence of the PI3KCA genes PCR amplified and sequenced toassess the activities of the fusion proteins in human cell culture.

It will be understood that the example of correcting point mutations inPI3KCA is provided for illustration purposes and is not meant to limitthe instant disclosure. The skilled artisan will understand that theinstantly disclosed DNA-editing fusion proteins can be used to correctother point mutations and mutations associated with other cancers andwith diseases other than cancer including other proliferative diseases.

The successful correction of point mutations in disease-associated genesand alleles opens up new strategies for gene correction withapplications in therapeutics and basic research. Site-specificsingle-base modification systems like the disclosed fusions of a Gamprotein, a napDNAbp and a cytidine deaminase domain also haveapplications in “reverse” gene therapy, where certain gene functions arepurposely suppressed or abolished. In these cases, site-specificallymutating Trp (TGG), Gln (CAA and CAG), or Arg (CGA) residues topremature stop codons (TAA, TAG, TGA) can be used to abolish proteinfunction in vitro, ex vivo, or in vivo.

The instant disclosure provides methods for the treatment of a subjectdiagnosed with a disease associated with or caused by a point mutationthat can be corrected by a base editing fusion protein provided herein.For example, in some embodiments, a method is provided that comprisesadministering to a subject having such a disease, e.g., a cancerassociated with a PI3KCA point mutation as described above, an effectiveamount of a base editor fusion protein that corrects the point mutationor introduces a deactivating mutation into the disease-associated gene.In some embodiments, the disease is a proliferative disease. In someembodiments, the disease is a genetic disease. In some embodiments, thedisease is a neoplastic disease. In some embodiments, the disease is ametabolic disease. In some embodiments, the disease is a lysosomalstorage disease. Other diseases that can be treated by correcting apoint mutation or introducing a deactivating mutation into adisease-associated gene will be known to those of skill in the art, andthe disclosure is not limited in this respect.

The instant disclosure provides methods for the treatment of additionaldiseases or disorders, e.g., diseases or disorders that are associatedor caused by a point mutation that can be corrected bydeaminase-mediated gene editing. Some such diseases are describedherein, and additional suitable diseases that can be treated with thestrategies and fusion proteins provided herein will be apparent to thoseof skill in the art based on the instant disclosure. Exemplary suitablediseases and disorders are listed below. It will be understood that thenumbering of the specific positions or residues in the respectivesequences depends on the particular protein and numbering scheme used.Numbering might be different, e.g., in precursors of a mature proteinand the mature protein itself, and differences in sequences from speciesto species may affect numbering. One of skill in the art will be able toidentify the respective residue in any homologous protein and in therespective encoding nucleic acid by methods well known in the art, e.g.,by sequence alignment and determination of homologous residues.Exemplary suitable diseases and disorders include, without limitation,cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR byCRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosispatients. Cell stem cell. 2013; 13: 653-658; and Wu et. al., Correctionof a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell.2013; 13: 659-662, neither of which uses a deaminase fusion protein tocorrect the genetic defect); phenylketonuria—e.g., phenylalanine toserine mutation at position 835 (mouse) or 240 (human) or a homologousresidue in phenylalanine hydroxylase gene (T>C mutation)—see, e.g.,McDonald et al., Genomics. 1997; 39:402-405; Bernard-Soulier syndrome(BSS)—e.g., phenylalanine to serine mutation at position 55 or ahomologous residue, or cysteine to arginine at residue 24 or ahomologous residue in the platelet membrane glycoprotein IX (T>Cmutation)—see, e.g., Noris et al., British Journal of Haematology. 1997;97: 312-320, and Ali et al., Hematol. 2014; 93: 381-384; epidermolytichyperkeratosis (EHK)—e.g., leucine to proline mutation at position 160or 161 (if counting the initiator methionine) or a homologous residue inkeratin 1 (T>C mutation)—see, e.g., Chipev et al., Cell. 1992; 70:821-828, see also accession number P04264 in the UNIPROT database atwww[dot]uniprot[dot]org; chronic obstructive pulmonary disease(COPD)—e.g., leucine to proline mutation at position 54 or 55 (ifcounting the initiator methionine) or a homologous residue in theprocessed form of α₁-antitrypsin or residue 78 in the unprocessed formor a homologous residue (T>C mutation)—see, e.g., Poller et al.,Genomics. 1993; 17: 740-743, see also accession number P01011 in theUNIPROT database; Charcot-Marie-Toot disease type 4J—e.g., isoleucine tothreonine mutation at position 41 or a homologous residue in FIG. 4 (T>Cmutation)—see, e.g., Lenk et al., PLoS Genetics. 2011; 7: e1002104;neuroblastoma (NB)—e.g., leucine to proline mutation at position 197 ora homologous residue in Caspase-9 (T>C mutation)—see, e.g., Kundu etal., 3 Biotech. 2013, 3:225-234; von Willebrand disease (vWD)—e.g.,cysteine to arginine mutation at position 509 or a homologous residue inthe processed form of von Willebrand factor, or at position 1272 or ahomologous residue in the unprocessed form of von Willebrand factor (T>Cmutation)—see, e.g., Lavergne et al., Br. J. Haematol. 1992, see alsoaccession number P04275 in the UNIPROT database; 82: 66-72; myotoniacongenital—e.g., cysteine to arginine mutation at position 277 or ahomologous residue in the muscle chloride channel gene CLCN1 (T>Cmutation)—see, e.g., Weinberger et al., The J. of Physiology. 2012; 590:3449-3464; hereditary renal amyloidosis—e.g., stop codon to argininemutation at position 78 or a homologous residue in the processed form ofapolipoprotein All or at position 101 or a homologous residue in theunprocessed form (T>C mutation)—see, e.g., Yazaki et al., Kidney Int.2003; 64: 11-16; dilated cardiomyopathy (DCM)—e.g., tryptophan toArginine mutation at position 148 or a homologous residue in the FOXD4gene (T>C mutation), see, e.g., Minoretti et. al., Int. J. of Mol. Med.2007; 19: 369-372; hereditary lymphedema—e.g., histidine to argininemutation at position 1035 or a homologous residue in VEGFR3 tyrosinekinase (A>G mutation), see, e.g., Irrthum et al., Am. J. Hum. Genet.2000; 67: 295-301; familial Alzheimer's disease—e.g., isoleucine tovaline mutation at position 143 or a homologous residue in presenilinl(A>G mutation), see, e.g., Gallo et. al., J. Alzheimer's disease. 2011;25: 425-431; Prion disease—e.g., methionine to valine mutation atposition 129 or a homologous residue in prion protein (A>Gmutation)—see, e.g., Lewis et. al., J. of General Virology. 2006; 87:2443-2449; chronic infantile neurologic cutaneous articular syndrome(CINCA)—e.g., Tyrosine to Cysteine mutation at position 570 or ahomologous residue in cryopyrin (A>G mutation)—see, e.g., Fujisawa et.al. Blood. 2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g.,arginine to glycine mutation at position 120 or a homologous residue inαβ crystallin (A>G mutation)—see, e.g., Kumar et al., J. Biol. Chem.1999; 274: 24137-24141. The entire contents of all references anddatabase entries is incorporated herein by reference.

It will be apparent to those of skill in the art that in order to targeta base editor fusion protein as disclosed herein to a target site, e.g.,a site comprising a point mutation to be edited, it is typicallynecessary to co-express the fusion protein together with a guide RNA,e.g., an sgRNA. As explained in more detail elsewhere herein, a guideRNA typically comprises a tracrRNA framework allowing for Cas9 binding,and a guide sequence, which confers sequence specificity to theCas9:nucleic acid editing enzyme/domain fusion protein. In someembodiments, the guide RNA comprises a structure 5′-[guidesequence]-guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3′ (SEQ ID NO: 398), wherein the guide sequence comprises a sequencethat is complementary to the target sequence. The guide sequence istypically 20 nucleotides long. The sequences of suitable guide RNAs fortargeting Cas9 fusion proteins to specific genomic target sites will beapparent to those of skill in the art based on the instant disclosure.Such suitable guide RNA sequences typically comprise guide sequencesthat are complementary to a nucleic sequence within 50 nucleotidesupstream or downstream of the target nucleotide to be edited. Someexemplary guide RNA sequences suitable for targeting Cas9 fusionproteins to specific target sequences are provided below. Exemplaryguide RNA structures, including guide RNA backbone sequences, aredescribed, for example, in Jinek M, et al. (2012) A programmabledual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science, 337, 816-812; Mali P, et al. (2013) Cas9 as a versatile toolfor engineering biology. Nature Methods, 10, 957-963; Li J F, et al.(2013) Multiplex and homologous recombination-mediated genome editing inArabidopsis and Nicotiana benthamiana using guide RNA and Cas9. NatureBiotech, 31, 688-691; Hwang W Y, et al. (2013) Efficient in vivo genomeediting using RNA-guided nucleases. Nat Biotechnol, 31, 227-229; Cong L,et al. (2013) Multiplex genome engineering using CRIPSR/Cas systems.Science, 339, 819-823; Cho S W, et al. (2013) Targeted genomeengineering in human cells with the Cas9 RNA-guided endonuclease. NatBiotechnol, 31, 230-232; Jinek M J, et al. (2013) RNA-programmed genomeediting in human cells. eLIFE, 2:e00471; DiCarlo J E, et al. (2013)Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.Nucl Acids Res, 41, 4336-4343; Qi L S, et al. (2013) Repruposing CRISPRas an RNA-Guided Platform for Sequence-Specific Control of GeneExpression. Cell, 152, 1173-1183; and Briner A E, et al. (2014) GuideRNA functional modules direct Cas9 activity and orthogonality. Mol Cell,56, 333-339; each of which is incorporated herein by reference.

Base Editor Efficiency

Some aspects of the disclosure are based on the recognition that any ofthe base editors provided herein are capable of modifying a specificnucleotide base without generating a significant proportion of indels.An “indel”, as used herein, refers to the insertion or deletion of anucleotide base within a nucleic acid. Such insertions or deletions canlead to frame shift mutations within a coding region of a gene. In someembodiments, it is desirable to generate base editors that efficientlymodify (e.g. mutate or deaminate) a specific nucleotide within a nucleicacid, without generating a large number of insertions or deletions(i.e., indels) in the nucleic acid. In certain embodiments, any of thebase editors provided herein are capable of generating a greaterproportion of intended modifications (e.g., point mutations ordeaminations) versus indels. In some embodiments, the base editorsprovided herein are capable of generating a ratio of intended pointmutations to indels that is greater than 1:1. In some embodiments, thebase editors provided herein are capable of generating a ratio ofintended point mutations to indels that is at least 1.5:1, at least 2:1,at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, atleast 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1,at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, atleast 400:1, at least 500:1, at least 600:1, at least 700:1, at least800:1, at least 900:1, or at least 1000:1, or more. The number ofintended mutations and indels may be determined using any suitablemethod, for example the methods used in the below Examples.

In some embodiments, the base editors provided herein are capable oflimiting formation of indels in a region of a nucleic acid. In someembodiments, the region is at a nucleotide targeted by a base editor ora region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of anucleotide targeted by a base editor. In some embodiments, any of thebase editors provided herein are capable of limiting the formation ofindels at a region of a nucleic acid to less than 1%, less than 1.5%,less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than8%, less than 9%, less than 10%, less than 12%, less than 15%, or lessthan 20%. The number of indels formed at a nucleic acid region maydepend on the amount of time a nucleic acid (e.g., a nucleic acid withinthe genome of a cell) is exposed to a base editor. In some embodiments,an number or proportion of indels is determined after at least 1 hour,at least 2 hours, at least 6 hours, at least 12 hours, at least 24hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4days, at least 5 days, at least 7 days, at least 10 days, or at least 14days of exposing a nucleic acid (e.g., a nucleic acid within the genomeof a cell) to a base editor.

Some aspects of the disclosure are based on the recognition that any ofthe base editors provided herein are capable of efficiently generatingan intended mutation, such as a point mutation, in a nucleic acid (e.g.a nucleic acid within a genome of a subject) without generating asignificant number of unintended mutations, such as unintended pointmutations. In some embodiments, a intended mutation is a mutation thatis generated by a specific base editor bound to a gRNA, specificallydesigned to generate the intended mutation. In some embodiments, theintended mutation is a mutation associated with a disease or disorder.In some embodiments, the intended mutation is a cytosine (C) to thymine(T) point mutation associated with a disease or disorder. In someembodiments, the intended mutation is a guanine (G) to adenine (A) pointmutation associated with a disease or disorder. In some embodiments, theintended mutation is a cytosine (C) to thymine (T) point mutation withinthe coding region of a gene. In some embodiments, the intended mutationis a guanine (G) to adenine (A) point mutation within the coding regionof a gene. In some embodiments, the intended mutation is a pointmutation that generates a stop codon, for example, a premature stopcodon within the coding region of a gene. In some embodiments, theintended mutation is a mutation that eliminates a stop codon. In someembodiments, the intended mutation is a mutation that alters thesplicing of a gene. In some embodiments, the intended mutation is amutation that alters the regulatory sequence of a gene (e.g., a genepromotor or gene repressor). In some embodiments, any of the baseeditors provided herein are capable of generating a ratio of intendedmutations to unintended mutations (e.g., intended pointmutations:unintended point mutations) that is greater than 1:1. In someembodiments, any of the base editors provided herein are capable ofgenerating a ratio of intended mutations to unintended mutations (e.g.,intended point mutations:unintended point mutations) that is at least1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, atleast 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1,at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, atleast 30:1, at least 40:1, at least 50:1, at least 100:1, at least150:1, at least 200:1, at least 250:1, at least 500:1, or at least1000:1, or more. It should be appreciated that the characterstics of thebase editors described in the “Base Editor Efficiency” section, herein,may be applied to any of the fusion proteins, or methods of using thefusion proteins provided herein.

Methods for Editing Nucleic Acids

Some aspects of the disclosure provide methods for editing a nucleicacid. In some embodiments, the method is a method for editing anucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNAsequence). In some embodiments, the method comprises the steps of: a)contacting a target region of a nucleic acid (e.g., a double-strandedDNA sequence) with a complex comprising a base editor (e.g., any of thefusion proteins provided herein) and a guide nucleic acid (e.g., gRNA),wherein the target region comprises a targeted nucleobase pair, b)inducing strand separation of said target region, c) converting a firstnucleobase of said target nucleobase pair in a single strand of thetarget region to a second nucleobase, and d) cutting no more than onestrand of said target region, where a third nucleobase complementary tothe first nucleobase base is replaced by a fourth nucleobasecomplementary to the second nucleobase; and the method results in lessthan 20% (e.g., less than 15%, 10%, 5%, 1%, 0.5% or 0.1%) indelformation in the nucleic acid. It should be appreciated that in someembodiments, step b is omitted. In some embodiments, the firstnucleobase is a cytosine. In some embodiments, the second nucleobase isa deaminated cytosine, or a uracil. In some embodiments, the thirdnucleobase is a guanine. In some embodiments, the fourth nucleobase isan adenine. In some embodiments, the first nucleobase is a cytosine, thesecond nucleobase is a deaminated cytosine, or a uracil, the thirdnucleobase is a guanine, and the fourth nucleobase is an adenine. Insome embodiments, the method results in less than 19%, 18%, 16%, 14%,12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indelformation. In some embodiments, the method further comprises replacingthe second nucleobase with a fifth nucleobase that is complementary tothe fourth nucleobase, thereby generating an intended edited base pair(e.g., C:G ->T:A). In some embodiments, the fifth nucleobase is athymine. In some embodiments, at least 5% of the intended basepaires areedited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, or 50% of the intended basepaires are edited.

In some embodiments, the ratio of intended products to unintendedproducts in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1,30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. Insome embodiments, the ratio of intended point mutation to indelformation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, ormore. In some embodiments, the cut single strand (nicked strand) ishybridized to the guide nucleic acid. In some embodiments, the cutsingle strand is opposite to the strand comprising the first nucleobase.In some embodiments, the base editor comprises a Cas9 domain. In someembodiments, the first base is cytosine, and the second base is not a G,C, A, or T. In some embodiments, the second base is uracil. In someembodiments, the first base is cytosine. In some embodiments, the secondbase is not a G, C, A, or T. In some embodiments, the second base isuracil. In some embodiments, the base editor inhibits base escisionrepair of the edited strand. In some embodiments, the base editorprotects or binds the non-edited strand. In some embodiments, the baseeditor comprises UGI activity. In some embodiments, the base editorcomprises nickase activity. In some embodiments, the intended editedbasepair is upstream of a PAM site. In some embodiments, the intendededited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In someembodiments, the intended edited basepair is downstream of a PAM site.In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesdownstream stream of the PAM site. In some embodiments, the method doesnot require a canonical (e.g., NGG) PAM site. In some embodiments, thenucleobase editor comprises a linker. In some embodiments, the linker is1-25 amino acids in length. In some embodiments, the linker is 5-20amino acids in length. In some embodiments, linker is 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acids in length. In someembodiments, the target region comprises a target window, wherein thetarget window comprises the target nucleobase pair. In some embodiments,the target window comprises 1-10 nucleotides. In some embodiments, thetarget window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1nucleotides in length. In some embodiments, the target window is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleotides in length. In some embodiments, the intended edited basepair is within the target window. In some embodiments, the target windowcomprises the intended edited base pair. In some embodiments, the methodis performed using any of the base editors provided herein. In someembodiments, a target window is a deamination window

In some embodiments, the disclosure provides methods for editing anucleotide. In some embodiments, the disclosure provides a method forediting a nucleobase pair of a double-stranded DNA sequence. In someembodiments, the method comprises a) contacting a target region of thedouble-stranded DNA sequence with a complex comprising a base editor anda guide nucleic acid (e.g., gRNA), where the target region comprises atarget nucleobase pair, b) inducing strand separation of said targetregion, c) converting a first nucleobase of said target nucleobase pairin a single strand of the target region to a second nucleobase, d)cutting no more than one strand of said target region, wherein a thirdnucleobase complementary to the first nucleobase base is replaced by afourth nucleobase complementary to the second nucleobase, and the secondnucleobase is replaced with a fifth nucleobase that is complementary tothe fourth nucleobase, thereby generating an intended edited basepair,wherein the efficiency of generating the intended edited basepair is atleast 5%. It should be appreciated that in some embodiments, step b isomitted. In some embodiments, at least 5% of the intended basepaires areedited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, or 50% of the intended basepaires are edited. In some embodiments,the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%,2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In someembodiments, the ratio of intended product to unintended products at thetarget nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1,60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments,the ratio of intended point mutation to indel formation is greater than1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments,the cut single strand is hybridized to the guide nucleic acid. In someembodiments, the cut single strand is opposite to the strand comprisingthe first nucleobase. In some embodiments, the first base is cytosine.In some embodiments, the second nucleobase is not G, C, A, or T. In someembodiments, the second base is uracil. In some embodiments, the baseeditor inhibits base escision repair of the edited strand. In someembodiments, the base editor protects or binds the non-edited strand. Insome embodiments, the nucleobase editor comprises UGI activity. In someembodiments, the nucleobase edit comprises nickase activity. In someembodiments, the intended edited basepair is upstream of a PAM site. Insome embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstreamof the PAM site. In some embodiments, the intended edited basepair isdownstream of a PAM site. In some embodiments, the intended edited basepair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 nucleotides downstream stream of the PAM site. In someembodiments, the method does not require a canonical (e.g., NGG) PAMsite. In some embodiments, the nucleobase editor comprises a linker. Insome embodiments, the linker is 1-25 amino acids in length. In someembodiments, the linker is 5-20 amino acids in length. In someembodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20amino acids in length. In some embodiments, the target region comprisesa target window, wherein the target window comprises the targetnucleobase pair. In some embodiments, the target window comprises 1-10nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7,1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In someembodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In someembodiments, the intended edited base pair occurs within the targetwindow. In some embodiments, the target window comprises the intendededited base pair. In some embodiments, the nucleobase editor is any oneof the base editors provided herein.

Pharmaceutical Compositions

In some embodiments, any of the fusion proteins, gRNAs, and/or complexesdescribed herein are provided as part of a pharmaceutical composition.In some embodiments, the pharmaceutical composition comprises any of thefusion proteins provided herein. In some embodiments, the pharmaceuticalcomposition comprises any of the complexes provided herein. In someembodiments, the pharmaceutical composition comprises aribonucleoprotein complex comprising an RNA-guided nuclease (e.g., Cas9)that forms a complex with a gRNA and a cationic lipid. In someembodiments pharmaceutical composition comprises a gRNA, a nucleic acidprogrammable DNA binding protein, a cationic lipid, and apharmaceutically acceptable excipient. Pharmaceutical compositions mayoptionally comprise one or more additional therapeutically activesubstances.

In some embodiments, compositions provided herein are administered to asubject, for example, to a human subject, in order to effect a targetedgenomic modification within the subject. In some embodiments, cells areobtained from the subject and contacted with a any of the pharmaceuticalcompositions provided herein. In some embodiments, cells removed from asubject and contacted ex vivo with a pharmaceutical composition arere-introduced into the subject, optionally after the desired genomicmodification has been effected or detected in the cells. Methods ofdelivering pharmaceutical compositions comprising nucleases are known,and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717;6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113;6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which areincorporated by reference herein in their entireties. Although thedescriptions of pharmaceutical compositions provided herein areprincipally directed to pharmaceutical compositions which are suitablefor administration to humans, it will be understood by the skilledartisan that such compositions are generally suitable for administrationto animals or organisms of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions is contemplated include, but are not limited to, humansand/or other primates; mammals, domesticated animals, pets, andcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,dogs, mice, and/or rats; and/or birds, including commercially relevantbirds such as chickens, ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient(s) into association with an excipientand/or one or more other accessory ingredients, and then, if necessaryand/or desirable, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated in its entirety herein by reference) disclosesvarious excipients used in formulating pharmaceutical compositions andknown techniques for the preparation thereof. See also PCT applicationPCT/US2010/055131 (Publication number WO2011053982 A8, filed Nov. 2,2010), incorporated in its entirety herein by reference, for additionalsuitable methods, reagents, excipients and solvents for producingpharmaceutical compositions comprising a nuclease. Except insofar as anyconventional excipient medium is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this disclosure.

In some embodiments, compositions in accordance with the presentinvention may be used for treatment of any of a variety of diseases,disorders, and/or conditions, including but not limited to one or moreof the following: autoimmune disorders (e.g. diabetes, lupus, multiplesclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders(e.g. arthritis, pelvic inflammatory disease); infectious diseases (e.g.viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungalinfections, sepsis); neurological disorders (e.g. Alzheimer's disease,Huntington's disease; autism; Duchenne muscular dystrophy);cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia,thrombosis, clotting disorders, angiogenic disorders such as maculardegeneration); proliferative disorders (e.g. cancer, benign neoplasms);respiratory disorders (e.g. chronic obstructive pulmonary disease);digestive disorders (e.g. inflammatory bowel disease, ulcers);musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine,metabolic, and nutritional disorders (e.g. diabetes, osteoporosis);urological disorders (e.g. renal disease); psychological disorders (e.g.depression, schizophrenia); skin disorders (e.g. wounds, eczema); bloodand lymphatic disorders (e.g. anemia, hemophilia); etc.

Kits, Vectors, Cells

Some aspects of this disclosure provide kits comprising a nucleic acidconstruct, comprising (a) a nucleotide sequence encoding any of thefusion proteins provided herein; and (b) a heterologous promoter thatdrives expression of the sequence of (a). In some embodiments, the kitfurther comprises an expression construct encoding a guide RNA backbone,wherein the construct comprises a cloning site positioned to allow thecloning of a nucleic acid sequence identical or complementary to atarget sequence into the guide RNA backbone.

Some aspects of this disclosure provide polynucleotides encoding afusion protein (e.g., base editor) as provided herein. Some aspects ofthis disclosure provide vectors comprising such polynucleotides. In someembodiments, the vector comprises a heterologous promoter drivingexpression of polynucleotide.

Some aspects of this disclosure provide cells comprising a any of thefusion proteins provided herein, a nucleic acid molecule encoding any ofthe fusion proteins provided herein, a complex comprising any of thefusion proteins provided herein and the gRNA, and/or a vector asprovided herein.

The description of exemplary embodiments of the reporter systems aboveis provided for illustration purposes only and not meant to be limiting.Additional variations of the exemplary compostions and methods describedin detail above, are also embraced by this disclosure.

REFERENCES

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Modified CRISPR/Cas9 System. Mol Plant 10, 526-529 (2017).

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CRISPR/Cas9 System. Mol Plant 10, 523-525 (2017).

-   8. Y. Ma et al., Targeted AID-mediated mutagenesis (TAM) enables    efficient genomic diversification in mammalian cells. Nat Methods    13, 1029-1035 (2016).-   9. K. Nishida et al., Targeted nucleotide editing using hybrid    prokaryotic and vertebrate adaptive immune systems. Science 353,    (2016).-   10. H. A. Rees et al., Improving the DNA specificity and    applicability of base editing through protein engineering and    protein delivery. Nat Commun 8, 15790 (2017).-   11. L. Yang et al., Engineering and optimising deaminase fusions for    genome editing. Nat

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-   12. Y. Zong et al., Precise base editing in rice, wheat and maize    with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35, 438-440    (2017).-   13. Z. Shimatani et al., Targeted base editing in rice and tomato    using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotech 35,    441-443 (2017).-   14. A. C. Komor, A. H. Badran, D. R. Liu, CRISPR-Based Technologies    for the Manipulation of Eukaryotic Genomes. Cell 168, 20-36 (2017).-   15. A. J. Davis, D. J. Chen, DNA double strand break repair via    non-homologous end-joining. Translational cancer research 2, 130-143    (2013).-   16. M. M. Vilenchik, A. G. Knudson, Endogenous DNA double-strand    breaks: Production, fidelity of repair, and induction of cancer.    Proceedings of the National Academy of Sciences 100, 12871-12876    (2003).-   17. F. Liang, M. Han, P. J. Romanienko, M. Jasin, Homology-directed    repair is a major doublestrand break repair pathway in mammalian    cells. Proceedings of the National Academy of Sciences 95, 5172-5177    (1998).-   18. Y. Miyaoka et al., Systematic quantification of HDR and NHEJ    reveals effects of locus, nuclease, and cell type on genome-editing.    Scientific Reports 6, 23549 (2016).-   19. M. M. Jore et al., Structural basis for CRISPR RNA-guided DNA    recognition by Cascade. Nat Struct Mol Biol 18, 529-536 (2011).-   20. L. H. Pearl, Structure and function in the uracil-DNA    glycosylase superfamily. Mutation Research/DNA Repair 460, 165-181    (2000).-   21. W. Tang, J. H. Hu, D. R. Liu, Aptazyme-embedded guide RNAs    enable ligand-responsive genome editing and transcriptional    activation. 8, 15939 (2017).-   22. G. Saraconi, F. Severi, C. Sala, G. Mattiuz, S. G. Conticello,    The RNA editing enzyme APOBEC1 induces somatic mutations and a    compatible mutational signature is present in esophageal    adenocarcinomas. Genome Biology 15, 417 (2014).-   23. R. M. Kohli et al., Local Sequence Targeting in the AID/APOBEC    Family Differentially Impacts Retroviral Restriction and Antibody    Diversification. Journal of Biological Chemistry 285, 40956-40964    (2010).-   24. L. Chelico, P. Pham, M. F. Goodman, Stochastic properties of    processive cytidine DNA deaminases AID and APOBEC3G. Philosophical    Transactions of the Royal Society B: Biological Sciences 364,    583-593 (2009).-   25. E. A. Kouzminova, A. Kuzminov, Patterns of chromosomal    fragmentation due to uracil-DNA incorporation reveal a novel    mechanism of replication-dependent double-stranded breaks. Molecular    Microbiology 68, 202-215 (2008).-   26. F. A. Ran et al., In vivo genome editing using Staphylococcus    aureus Cas9. Nature 520, 186-191 (2015).-   27. F. d. A. di Fagagna, G. R. Weller, A. J. Doherty, S. P. Jackson,    The Gam protein of bacteriophage Mu is an orthologue of eukaryotic    Ku. EMBO Reports 4, 47-52 (2003).-   28. C. Shee et al., Engineered proteins detect spontaneous DNA    breakage in human and bacterial cells. eLife 2, e01222 (2013).

Example 2: Exemplary Cas9 Sequences from Various Species

This disclosure provides Cas9 variants in which one or more of the aminoacid residues identified (e.g., by an asterisk) are mutated as describedherein. In some embodiments, the D10 and H840 residues are mutated,e.g., to an alanine residue, and the Cas9 variants provided include oneor more additional mutations of the amino acid residues identified by anasterisk as provided herein. In some embodiments, the D10 residue ismutated, e.g., to an alanine residue, and the Cas9 variants providedinclude one or more additional mutations of the amino acid residues(e.g., identified by an asterisk) as provided herein.

A number of Cas9 sequences from various species were aligned todetermine whether corresponding homologous amino acid residues can beidentified in other Cas9 domains, allowing the generation of Cas9variants with corresponding mutations of the homologous amino acidresidues. The alignment was carried out using the NCBI Constraint-basedMultiple Alignment Tool (COBALT(accessible atst-va.ncbi.nlm.nih.gov/tools/cobalt), with the following parameters.Alignment parameters: Gap penalties -11,-1; End-Gap penalties -5,-1. CDDParameters: Use RPS BLAST on; Blast E-value 0.003; Find Conservedcolumns and Recompute on. Query Clustering Parameters: Use queryclusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular.

An exemplary alignment of four Cas9 sequences is provided below. TheCas9 sequences in the alignment are: Sequence 1 (S1): SEQ ID NO: 11 IWP_010922251 I gi 499224711 I type II CRISPR RNA-guided endonucleaseCas9 [Streptococcus pyogenes]; Sequence 2 (S2): SEQ ID NO: 12 IWP_039695303 I gi 746743737 I type II CRISPR RNA-guided endonucleaseCas9 [Streptococcus gallolyticus]; Sequence 3 (S3): SEQ ID NO: 13WP_045635197 I gi 782887988 I type II CRISPR RNA-guided endonucleaseCas9 [Streptococcus mitis]; Sequence 4 (S4): SEQ ID NO: 14 I SAXW_A I gi924443546 I Staphylococcus Aureus Cas9. The HNH domain (bold andunderlined) and the RuvC domain (boxed) are identified for each of thefour sequences. Amino acid residues 10, 122, 137, 182, 262, 294, 409,480, 543, 660, 694, 840, 1219, and 1329 in Si and the homologous aminoacids in the aligned sequences are identified with an asterisk followingthe respective amino acid residue. A similar approach can be employed todetermine homologous amino acid residues suitable for mutation based onthe amino acid mutations of Cas9 domains identified herein.

S1 1 --MDKK- YSIGLD*IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI--GALLFDSG--ETAEATRLKRTARRRYT   73 S2 1 --MTKKNYSIGLD*IGTNSVGWAVITDDYKVPAKKMKVLGNTDKKYIKKNLL--GALLFDSG--ETAEATRLKRTARRRYT   74 S3 1 --M-KKGYSIGLD*IGTNSVGFAVITDDYKVPSKKMKVLGNTDKRFIKKNLI--GALLFDEG--TTAEARRLKRTARRRYT   73 S4 1 GSHMKRNYILGLD*IGITSVGYGII--DYET-----------------RDVIDAGVRLFKEANVENNEGRRSKRGARRLKR   61 S1 74RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI*VDEVAYHEKYPTIYH*LRKKLVDSTDKADLRL 153 S2 75RRKNRLRYLQEIFANEIAKVDESFFQRLDESFLTDDDKTFDSHPIFGNK*AEEDAYHQKFPTIYH*LRKHLADSSEKADLRL 154 S3 74RRKNRLRYLQEIFSEEMSKVDSSFFHRLDDSFLIPEDKRESKYPIFATL*TEEKEYHKQFPTIYH*LRKQLADSKEKTDLRL 153 S4 62RRRHRIQRVKKLL--------------FDYNLLTD---------------------HSELSGINP*YEARVKGLSQKLSEEE 107 S1 154IYLALAHMIKFRGHFLIEGDLNPDNSDVD*KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK 233 S2 155VYLALAHMIKFRGHFLIEGELNAENTDVQ*KIFADFVGVYNRTFDDSHLSEITVDVASILTEKISKSRRLENLIKYYPTEK 234 S3 154IYLALAHMIKYRGHFLYEEAFDIKNNDIQ*KIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLFPDEK 233 S4 108FSAALLHLAKRRG-----------------------VHNVNEVEEDT---------------------------------- 131 S1 234KNGLFGNLIALSLGLTPNFKSNFDLAEDA*KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK*NLSDAILLSDILRVNTEIT 313 S2 235KNTLFGNLIALALGLQPNFKTNFKLSEDA*KLQFSKDTYEEDLEELLGKIGDDYADLFTSAK*NLYDAILLSGILTVDDNST 314 S3 234STGLFSEFLKLIVGNQADFKKHFDLEDKA*PLQFSKDTYDEDLENLLGQIGDDFTDLFVSAK*KLYDAILLSGILTVTDPST 313 S4 132-----GNELS------------------T*KEQISRN--------------------------------------------- 144 S1 314KAPLSASMIKRYDEHHQDLITLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM--DGTEELLV 391 S2 315KAPLSASMIKRYVEHHEDLEKLKEFIKANKSELYHDIFKDKNKNGYAGYIENGVKQDEFYKYLKNILSKIKIDGSDYFLD 394 S3 314KAPLSASMIERYENHQNDLAALKQFIKNNLPEKYDEVFSDQSKDGYAGYIDGKTTQETFYKYIKNLLSKF--EGTDYFLD 391 S4 145----SKALEEKYVAELQ-------------------------------------------------LERLKKDG------ 165 S1 392KLNREDLLRKQRTFDNGS*IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE 471 S2 395KIEREDFLRKQRTFDNGS*IPHQIHLQEMHAILRRQGDYYPFLKEKQDRIEKILTFRIPYYVGPLVRKDSRFAWAEYRSDE 474 S3 392KIEREDFLRKQRTFDNGS*IPHQIHLQEMNAILRRQGEYYPFLKDNKEKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDE 471 S4 166--EVRGSINRFKTSD---------YVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGP--GEGSPFGW------K 227 S1 472TITPWNFEE*VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE*QKKAIVDL 551 S2 475KITPWNFDK*VIDKEKSAEKFITRMTLNDLYLPEEKVLPKHSHVYETYAVYNELTKIKYVNEQGKE-SFFDSN*MKQEIFDH 553 S3 472AIRPWNFEE*IVDKASSAEDFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKVKFIAEGLRDYQFLDSG*QKKQIVNQ 551 S4 228DIKEW----------------YEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEK---LEYY*EKFQIIEN 289  S1 552LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR---FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 628 S2 554VFKENRKVTKEKLLNYLNKEFPEYRIKDLIGLDKENKSFNASLGTYHDLKKIL-DKAFLDDKVNEEVIEDIIKTLTLFED 632 S3 552LFKENRKVTEKDIIHYLHN-VDGYDGIELKGIEKQ---FNASLSTYHDLLKIIKDKEFMDDAKNEAILENIVHTLTIFED 627 S4 290VFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF---TNLKVYHDIKDITARKEII---ENAELLDQIAKILTIYQS 363 S1 629REMIEERLKTYAHLFDDKVMKQLKR-RRYTGWG*RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM*QLIHDDSLTFKED 707 S2 633KDMIHERLQKYSDIFTANQLKKLER-RHYTGWG*RLSYKLINGIRNKENNKTILDYLIDDGSANRNFM*QLINDDTLPFKQI 711 S3 628REMIKQRLAQYDSLFDEKVIKALTR-RHYTGWG*KLSAKLINGICDKQTGNTILDYLIDDGKINRNFM*QLINDDGLSFKEI 706 S4 364SEDIQEELTNLNSELTQEEIEQISNLKGYTGTH*NLSLKAINLILDE------LWHTNDNQIAIFNRL*KLVP--------- 428 S1 708

 781 S2 712

 784 S3 707

 779 S4 429

 505 S1 782KRIEEGIKELGSQIL-------KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD----YDVDH*IVPQSFLKDD 850 S2 785KKLQNSLKELGSNILNEEKPSYIEDKVENSHLQNDQLFLYYIQNGKDMYTGDELDIDHLSD----YDIDH*IIPQAFIKDD 860 S3 780KRIEDSLKILASGL---DSNILKENPTDNNQLQNDRLFLYYLQNGKDMYTGEALDINQLSS----YDIDH*IIPQAFIKDD 852 S4 506ERIEEIIRTTGK---------------ENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDH*IIPRSVSFDN 570 S1 851

 922 S2 861

 932 S3 853

 924 S4 571

 650 S1 923

1002 S2 933

1012 S3 925

1004 S4 651

 712 S1 1003

1077 S2 1013

1083 S3 1005

1081 S4 713

 764 S1 1078

1149 S2 1084

1158 S3 1082

1156 S4 765

 835 S1 1150EKGKSKKLKSVKELLGITIMERSSFEKNPI-DFLEAKG-----YKEVKKDLIIKLPKYSLFELENGRKRMLASAGE*LQKG1223 S2 1159EKGKAKKLKTVKELVGISIMERSFFEENPV-EFLENKG-----YHNIREDKLIKLPKYSLFEFEGGRRRLLASASE*LQKG1232 S3 1157EKGKAKKLKTVKTLVGITIMEKAAFEENPI-TFLENKG-----YHNVRKENILCLPKYSLFELENGRRRLLASAKE*LQKG1230 S4 836DPQTYQKLK--------LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS*RNKV 907 S1 1224NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH------1297 S2 1233NEMVLPGYLVELLYHAHRADNF-----NSTEYLNYVSEHKKEFEKVLSCVEDFANLYVDVEKNLSKIRAVADSM------1301 S3 1231NEIVLPVYLTTLLYHSKNVHKL-----DEPGHLEYIQKHRNEFKDLLNLVSEFSQKYVLADANLEKIKSLYADN------1299 S4 908VKLSLKPYRFD-VYLDNGVYKFV-----TVKNLDVIK--KENYYEVNSKAYEEAKKLKKISNQAEFIASFYNNDLIKING 979 S1 1298RDKPIREQAENIIHLFTLTNLGAPAAFKYFDT*TIDRKRYTSTKEVLDATLIHQSIT--------GLYETRI----DLSQL1365 S2 1302DNFSIEEISNSFINLLTLTALGAPADFNFLGE*KIPRKRYTSTKECLNATLIHQSIT--------GLYETRI----DLSKL1369 S3 1300EQADIEILANSFINLLTFTALGAPAAFKFFGK*DIDRKRYTTVSEILNATLIHQSIT--------GLYETWI----DLSKL1367 S4 980ELYRVIGVNNDLLNRIEVNMIDITYR-EYLEN*MNDKRPPRIIKTIASKT---QSIKKYSTDILGNLYEVKSKKHPQIIKK1055 S1 1366 GGD 1368 S2 1370 GEE 1372 S3 1368 GED 1370 S4 1056 G-- 1056

The alignment demonstrates that amino acid sequences and amino acidresidues that are homologous to a reference Cas9 amino acid sequence oramino acid residue can be identified across Cas9 sequence variants,including, but not limited to Cas9 sequences from different species, byidentifying the amino acid sequence or residue that aligns with thereference sequence or the reference residue using alignment programs andalgorithms known in the art. This disclosure provides Cas9 variants inwhich one or more of the amino acid residues identified by an asteriskin SEQ ID NO: 11 are mutated as described herein. The residues in Cas9sequences other than SEQ ID NO: 11 that correspond to the residuesidentified in SEQ ID NO: 11 by an asterisk are referred to herein as“homologous” or “corresponding” residues. Such homologous residues canbe identified by sequence alignment, e.g., as described above, and byidentifying the sequence or residue that aligns with the referencesequence or residue. Similarly, mutations in Cas9 sequences other thanSEQ ID NO: 11 that correspond to mutations identified in SEQ ID NO: 11herein, e.g., mutations of residues 10, 122, 137, 182, 262, 294, 409,480, 543, 660, 694, 840, 1219, and 1329 in SEQ ID NO: 11, are referredto herein as “homologous” or “corresponding” mutations. For example, themutations corresponding to the D10A mutation in 51 for the four alignedsequences above are D10A for S2, D9A for S3, and D13A for S4; thecorresponding mutations for H840A in 51 are H850A for S2, H842A for S3,and H560 for S4; the corresponding mutation for X1219V in 51 are X1228Vfor S2, X1226 for S3, and X903V for S4, and so on.

A total of 250 Cas9 sequences (SEQ ID NOs: 11-260) from differentspecies were aligned using the same algorithm and alignment parametersoutlined above, and is provided in Patent Publication No. WO2017/070633,published Apr. 27, 2017, entitled “Evolved Cas9 domains For GeneEditing”; the entire contents of which are incorporated herein byreference. Additional suitable Cas9 homologues, as well as performingalignments of homologues, will be apparent to those of ordinary skill inthe art based on this disclosure and knowledge in the field, and arewithin the scope of the present disclosure.

Cas9 variants with one or more mutations in amino acid residueshomologous to amino acid residues 122, 137, 182, 262, 294, 409, 480,543, 660, 694, 1219, and 1329 of SEQ ID NO: 11 are provided herein. Insome embodiments, the Cas9 variants provided herein comprise mutationscorresponding to the D10A and the H840A mutations in SEQ ID NO: 11,resulting in a nuclease-inactive dCas9, and at least one, at least two,at least three, at least four, at least five, at least six, or at leastseven mutations of amino acid residues homologous to amino acid residues122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of SEQID NO: 11.

Cas9 variants with one or more mutations in amino acid residueshomologous to amino acid residues 122, 137, 182, 262, 294, 409, 480,543, 660, 694, 1219, and 1329 of SEQ ID NO: 11 are provided herein. Insome embodiments, the Cas9 variants provided herein comprise mutationscorresponding to the D10A mutations in SEQ ID NO: 11, resulting in apartially nuclease-inactive dCas9, wherein the Cas9 can nick thenon-target strand but not the targeted strand, and at least one, atleast two, at least three, at least four, at least five, at least six,or at least seven mutations of amino acid residues homologous to aminoacid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219,and 1329 of SEQ ID NO: 11.

WP_010922251.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 11 WP_039695303.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus gallolyticus] SEQ ID NO: 12WP_045635197.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mitis] SEQ ID NO: 13 5AXW_A Cas9, Chain A, CrystalStructure [Staphylococcus Aureus] SEQ ID NO: 14 WP_009880683.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO:15 WP_010922251.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 16 WP_011054416.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 17WP_011284745.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 18 WP_011285506.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 19WP_011527619.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 20 WP_012560673.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 21WP_014407541.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 22 WP_020905136.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 23WP_023080005.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 24 WP_023610282.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 25WP_030125963.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 26 WP_030126706.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 27WP_031488318.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 28 WP_032460140.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 29WP_032461047.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 30 WP_032462016.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 31WP_032462936.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 32 WP_032464890.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 33WP_033888930.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 34 WP_038431314.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 35WP_038432938.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pyogenes] SEQ ID NO: 36 WP_038434062.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 37BAQ51233.1 CRISPR-associated protein, Csn1 family [Streptococcuspyogenes] SEQ ID NO: 38 KGE60162.1 hypothetical protein MGAS2111_0903[Streptococcus pyogenes SEQ ID NO: 39 MGAS2111] KGE60856.1CRISPR-associated endonuclease protein [Streptococcus pyogenes SS1447]SEQ ID NO: 40 WP_002989955.1 MULTISPECIES: type II CRISPR[Streptococcus] SEQ ID NO: 41 RNA-guided endonuclease Cas9WP_003030002.1 MULTISPECIES: type II CRISPR [Streptococcus] SEQ ID NO:42 RNA-guided endonuclease Cas9 WP_003065552.1 MULTISPECIES: type IICRISPR [Streptococcus] SEQ ID NO: 43 RNA-guided endonuclease Cas9WP_001040076.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 44 WP_001040078.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 45WP_001040080.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 46 WP_001040081.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 47WP_001040083.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 48 WP_001040085.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 49WP_001040087.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 50 WP_001040088.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 51WP_001040089.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 52 WP_001040090.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 53WP_001040091.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 54 WP_001040092.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 55WP_001040094.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 56 WP_001040095.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 57WP_001040096.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 58 WP_001040097.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 59WP_001040098.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 60 WP_001040099.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 61WP_001040100.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 62 WP_001040104.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 63WP_001040105.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 64 WP_001040106.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 65WP_001040107.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 66 WP_001040108.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 67WP_001040109.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 68 WP_001040110.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 69WP_015058523.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 70 WP_017643650.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 71WP_017647151.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 72 WP_017648376.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 73WP_017649527.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 74 WP_017771611.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 75WP_017771984.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 76 CFQ25032.1 CRISPR-associatedprotein [Streptococcus agalactiae] SEQ ID NO: 77 CFV16040.1CRISPR-associated protein [Streptococcus agalactiae] SEQ ID NO: 78KLJ37842.1 CRISPR-associated protein Csn1 [Streptococcus agalactiae] SEQID NO: 79 KLJ72361.1 CRISPR-associated protein Csn1 [Streptococcusagalactiae] SEQ ID NO: 80 KLL20707.1 CRISPR-associated protein Csn1[Streptococcus agalactiae] SEQ ID NO: 81 KLL42645.1 CRISPR-associatedprotein Csn1 [Streptococcus agalactiae] SEQ ID NO: 82 WP_047207273.1type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae]SEQ ID NO: 83 WP_047209694.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 84 WP_050198062.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 85WP_050201642.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 86 WP_050204027.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 87WP_050881965.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus agalactiae] SEQ ID NO: 88 WP_050886065.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 89AHN30376.1 CRISPR-associated protein Csn1 [Streptococcus agalactiae138P] SEQ ID NO: 90 EAO78426.1 reticulocyte binding protein[Streptococcus agalactiae H36B] SEQ ID NO: 91 CCW42055.1CRISPR-associated protein, SAG0894 family [Streptococcus agalactiaeILRI112] SEQ ID NO: 92 WP_003041502.1 type II CRISPR RNA-guidedendonuclease Cas9 [Streptococcus anginosus] SEQ ID NO: 93 WP_037593752.1type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus anginosus]SEQ ID NO: 94 WP_049516684.1 CRISPR-associated protein Csn1[Streptococcus anginosus] SEQ ID NO: 95 GAD46167.1 hypothetical proteinANG6_0662 [Streptococcus anginosus T5] SEQ ID NO: 96 WP_018363470.1 typeII CRISPR RNA-guided endonuclease Cas9 [Streptococcus caballi] SEQ IDNO: 97 WP_003043819.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus canis] SEQ ID NO: 98 WP_006269658.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus constellatus] SEQ ID NO: 99WP_048800889.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus constellatus] SEQ ID NO: 100 WP_012767106.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus dysgalactiae] SEQ IDNO: 101 WP_014612333.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus dysgalactiae] SEQ ID NO: 102 WP_015017095.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus dysgalactiae] SEQ IDNO: 103 WP_015057649.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus dysgalactiae] SEQ ID NO: 104 WP_048327215.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus dysgalactiae] SEQ IDNO: 105 WP_049519324.1 CRISPR-associated protein Csn1 [Streptococcusdysgalactiae] SEQ ID NO: 106 WP_012515931.1 type II CRISPR RNA-guidedendonuclease Cas9 [Streptococcus equi] SEQ ID NO: 107 WP_021320964.1type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus equi] SEQ IDNO: 108 WP_037581760.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus equi] SEQ ID NO: 109 WP _004232481.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus equinus] SEQ ID NO: 110WP_009854540.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus gallolyticus] SEQ ID NO: 111 WP_012962174.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus gallolyticus] SEQ IDNO: 112 WP_039695303.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus gallolyticus] SEQ ID NO: 113 WP_014334983.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus infantarius] SEQ IDNO: 114 WP_003099269.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus iniae] SEQ ID NO: 115 AHY15608.1 CRISPR-associatedprotein Csn1 [Streptococcus iniae] SEQ ID NO: 116 AHY17476.1CRISPR-associated protein Csn1 [Streptococcus iniae] SEQ ID NO: 117ESR09100.1 hypothetical protein IUSA1_08595 [Streptococcus iniae IUSA1]SEQ ID NO: 118 AGM98575.1 CRISPR-associated protein Cas9/Csn1,[Streptococcus iniae SF1] SEQ ID NO: 119 subtype II/NMEMI ALF27331.1CRISPR-associated protein Csn1 [Streptococcus intermedius] SEQ ID NO:120 WP_018372492.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus massiliensis] SEQ ID NO: 121 WP_045618028.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus mitis] SEQ ID NO: 122WP_045635197.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mitis] SEQ ID NO: 123 WP_002263549.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 124WP_002263887.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 125 WP_002264920.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 126WP_002269043.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 127 WP_002269448.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 128WP_002271977.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 129 WP_002272766.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 130WP_002273241.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 131 WP_002275430.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 132WP_002276448.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 133 WP_002277050.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 134WP_002277364.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 135 WP_002279025.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 136WP_002279859.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 137 WP_002280230.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 138WP_002281696.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 139 WP_002282247.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 140WP_002282906.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 141 WP_002283846.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 142WP_002287255.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 143 WP_002288990.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 144WP_002289641.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 145 WP_002290427.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 146WP_002295753.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 147 WP_002296423.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 148WP_002304487.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 149 WP_002305844.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 150WP_002307203.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 151 WP_002310390.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 152WP_002352408.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 153 WP_012997688.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 154WP_014677909.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 155 WP_019312892.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 156WP_019313659.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 157 WP_019314093.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 158WP_019315370.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 159 WP_019803776.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 160WP_019805234.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 161 WP_024783594.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 162WP_024784288.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 163 WP_024784666.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 164WP_024784894.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus mutans] SEQ ID NO: 165 WP_024786433.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus mutans] SEQ ID NO: 166WP_049473442.1 CRISPR-associated protein Csn1 [Streptococcus mutans] SEQID NO: 167 WP_049474547.1 CRISPR-associated protein Csn1 [Streptococcusmutans] SEQ ID NO: 168 EMC03581.1 hypothetical protein SMU69_09359[Streptococcus mutans NLML4] SEQ ID NO: 169 WP_000428612.1 type IICRISPR RNA-guided endonuclease Cas9 [Streptococcus oralis] SEQ ID NO:170 WP_000428613.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus oralis] SEQ ID NO: 171 WP_049523028.1 CRISPR-associatedprotein Csn1 [Streptococcus parasanguinis] SEQ ID NO: 172 WP_003107102.1type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus parauberis]SEQ ID NO: 173 WP_054279288.1 type II CRISPR RNA-guided endonucleaseCas9 [Streptococcus phocae] SEQ ID NO: 174 WP_049531101.1CRISPR-associated protein Csn1 [Streptococcus pseudopneumoniae] SEQ IDNO: 175 WP_049538452.1 CRISPR-associated protein Csn1 [Streptococcuspseudopneumoniae] SEQ ID NO: 176 WP_049549711.1 CRISPR-associatedprotein Csn1 [Streptococcus pseudopneumoniae] SEQ ID NO: 177WP_007896501.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus pseudoporcinus] SEQ ID NO: 178 EFR44625.1CRISPR-associated protein, Csn1 family [Streptococcus pseudoporcinus SEQID NO: 179 SPIN 20026] WP_002897477.1 type II CRISPR RNA-guidedendonuclease Cas9 [Streptococcus sanguinis] SEQ ID NO: 180WP_002906454.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus sanguinis] SEQ ID NO: 181 WP_009729476.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus sp. F0441] SEQ ID NO: 182CQR24647.1 CRISPR-associated protein [Streptococcus sp. FF10] SEQ ID NO:183 WP_000066813.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus sp. M334] SEQ ID NO: 184 WP_009754323.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus sp. taxon 056] SEQ ID NO:185 WP_044674937.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus suis] SEQ ID NO: 186 WP_044676715.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus suis] SEQ ID NO: 187WP_044680361.1 type II CRISPR RNA-guided endonuclease Cas9[Streptococcus suis] SEQ ID NO: 188 WP_044681799.1 type II CRISPRRNA-guided endonuclease Cas9 [Streptococcus suis] SEQ ID NO: 189WP_049533112.1 CRISPR-associated protein Csn1 [Streptococcus suis] SEQID NO: 190 WP_029090905.1 type II CRISPR RNA-guided endonuclease Cas9[Brochothrix thermosphacta] SEQ ID NO: 191 WP_006506696.1 type II CRISPRRNA-guided endonuclease Cas9 [Catenibacterium mitsuokai] SEQ ID NO: 192AIT42264.1 Cas9hc:NLS:HA [Cloning vector pYB196] SEQ ID NO: 193WP_034440723.1 type II CRISPR endonuclease Cas9 [Clostridiales bacteriumS5-A11] SEQ ID NO: 194 AKQ21048.1 Cas9 [CRISPR-mediated gene SEQ ID NO:195 targeting vector p (bhsp68-Cas9)] WP_004636532.1 type II CRISPRRNA-guided endonuclease Cas9 [Dolosigranulum pigrum] SEQ ID NO: 196WP_002364836.1 MULTISPECIES: type II CRISPR [Enterococcus] SEQ ID NO:197 RNA-guided endonuclease Cas9 WP_016631044.1 MULTISPECIES: type IICRISPR [Enterococcus] SEQ ID NO: 198 RNA-guided endonuclease Cas9EMS75795.1 hypothetical protein H318_06676 [Enterococcus durans IPLA6551] SEQ ID NO: 199 WP_002373311.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus faecalis] SEQ ID NO: 200 WP_002378009.1type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus faecalis] SEQID NO: 201 WP_002407324.1 type II CRISPR RNA-guided endonuclease Cas9[Enterococcus faecalis] SEQ ID NO: 202 WP_002413717.1 type II CRISPRRNA-guided endonuclease Cas9 [Enterococcus faecalis] SEQ ID NO: 203WP_010775580.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcusfaecalis] SEQ ID NO: 204 WP_010818269.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus faecalis] SEQ ID NO: 205 WP_010824395.1type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus faecalis] SEQID NO: 206 WP_016622645.1 type II CRISPR RNA-guided endonuclease Cas9[Enterococcus faecalis] SEQ ID NO: 207 WP_033624816.1 type II CRISPRRNA-guided endonuclease Cas9 [Enterococcus faecalis] SEQ ID NO: 208WP_033625576.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcusfaecalis] SEQ ID NO: 209 WP_033789179.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus faecalis] SEQ ID NO: 210 WP_002310644.1type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus faecium] SEQID NO: 211 WP_002312694.1 type II CRISPR RNA-guided endonuclease Cas9[Enterococcus faecium] SEQ ID NO: 212 WP_002314015.1 type II CRISPRRNA-guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 213WP_002320716.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcusfaecium] SEQ ID NO: 214 WP_002330729.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 215 WP_002335161.1type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus faecium] SEQID NO: 216 WP_002345439.1 type II CRISPR RNA-guided endonuclease Cas9[Enterococcus faecium] SEQ ID NO: 217 WP_034867970.1 type II CRISPRRNA-guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 218WP_047937432.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcusfaecium] SEQ ID NO: 219 WP_010720994.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus hirae] SEQ ID NO: 220 WP_010737004.1type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus hirae] SEQ IDNO: 221 WP_034700478.1 type II CRISPR RNA-guided endonuclease Cas9[Enterococcus hirae] SEQ ID NO: 222 WP_007209003.1 type II CRISPRRNA-guided endonuclease Cas9 [Enterococcus italicus] SEQ ID NO: 223WP_023519017.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcusmundtii] SEQ ID NO: 224 WP_010770040.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus phoeniculicola] SEQ ID NO: 225WP_048604708.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcussp. AM1] SEQ ID NO: 226 WP_010750235.1 type II CRISPR RNA-guidedendonuclease Cas9 [Enterococcus villorum] SEQ ID NO: 227 AII16583.1 Cas9endonuclease [Expression vector pCas9] SEQ ID NO: 228 WP_029073316.1type II CRISPR RNA-guided endonuclease Cas9 [Kandleria vitulina] SEQ IDNO: 229 WP_031589969.1 type II CRISPR RNA-guided endonuclease Cas9[Kandleria vitulina] SEQ ID NO: 230 KDA45870.1 CRISPR-associated proteinCas9/Csn1, [Lactobacillus animalis] SEQ ID NO: 231 subtype II/NMEMIWP_039099354.1 type II CRISPR RNA-guided endonuclease Cas9[Lactobacillus curvatus] SEQ ID NO: 232 AKP02966.1 hypothetical proteinABB45_04605 [Lactobacillus farciminis] SEQ ID NO: 233 WP_010991369.1type II CRISPR RNA-guided endonuclease Cas9 [Listeria innocua] SEQ IDNO: 234 WP_033838504.1 type II CRISPR RNA-guided endonuclease Cas9[Listeria innocua] SEQ ID NO: 235 EHN60060.1 CRISPR-associated protein,Csn1 family [Listeria innocua ATCC 33091] SEQ ID NO: 236 EFR89594.1crispr-associated protein, Csn1 family [Listeria innocua FSL S4-378] SEQID NO: 237 WP_038409211.1 type II CRISPR RNA-guided endonuclease Cas9[Listeria ivanovii] SEQ ID NO: 238 EFR95520.1 crispr-associated proteinCsn1 [Listeria ivanovii FSL F6-596] SEQ ID NO: 239 WP_003723650.1 typeII CRISPR RNA-guided endonuclease Cas9 [Listeria monocytogenes] SEQ IDNO: 240 WP_003727705.1 type II CRISPR RNA-guided endonuclease Cas9[Listeria monocytogenes] SEQ ID NO: 241 WP_003730785.1 type II CRISPRRNA-guided endonuclease Cas9 [Listeria monocytogenes] SEQ ID NO: 242WP_003733029.1 type II CRISPR RNA-guided endonuclease Cas9 [Listeriamonocytogenes] SEQ ID NO: 243 WP_003739838.1 type II CRISPR RNA-guidedendonuclease Cas9 [Listeria monocytogenes] SEQ ID NO: 244 WP_014601172.1type II CRISPR RNA-guided endonuclease Cas9 [Listeria monocytogenes] SEQID NO: 245 WP_023548323.1 type II CRISPR RNA-guided endonuclease Cas9[Listeria monocytogenes] SEQ ID NO: 246 WP_031665337.1 type II CRISPRRNA-guided endonuclease Cas9 [Listeria monocytogenes] SEQ ID NO: 247WP_031669209.1 type II CRISPR RNA-guided endonuclease Cas9 [Listeriamonocytogenes] SEQ ID NO: 248 WP_033920898.1 type II CRISPR RNA-guidedendonuclease Cas9 [Listeria monocytogenes] SEQ ID NO: 249 AKI42028.1CRISPR-associated protein [Listeria monocytogenes] SEQ ID NO: 250AKI50529.1 CRISPR-associated protein [Listeria monocytogenes] SEQ ID NO:251 EFR83390.1 crispr-associated protein Csn1 [Listeria monocytogenesSEQ ID NO: 252 FSL F2-208] WP_046323366.1 type II CRISPR RNA-guidedendonuclease Cas9 [Listeria seeligeri] SEQ ID NO: 253 AKE81011.1 Cas9[Plant multiplex genome editing SEQ ID NO: 254 vectorpYLCRISPR/Cas9Pubi-H] CUO82355.1 Uncharacterized protein conserved inbacteria [Roseburia hominis] SEQ ID NO: 255 WP_033162887.1 type IICRISPR RNA-guided endonuclease Cas9 [Sharpea azabuensis] SEQ ID NO: 256AGZ01981.1 Cas9 endonuclease [synthetic construct] SEQ ID NO: 257AKA60242.1 nuclease deficient Cas9 [synthetic construct] SEQ ID NO: 258AKS40380.1 Cas9 [Synthetic plasmid pFC330] SEQ ID NO: 259 4UN5_B Cas9,Chain B, Crystal Structure SEQ ID NO: 260

Additional suitable Cas9 sequences in which amino acid residueshomologous to residues 50, 86, 115, 108, 141, 175, 217, 230, 257, 261,262, 267, 274, 284, 294, 331, 319, 324, 341, 388, 405, 409, 435, 461,466, 480, 510, 522, 543, 548, 593, 653, 673, 694, 711, 712, 715, 772,777, 798, 811, 839, 847, 955, 967, 991, 1063, 1139, 1199, 1207, 1219,1224, 1227, 1229, 1256, 1264, 1296, 1318, 1356, and/or 1362 of SEQ IDNO: 11 can be identified are known to those of skill in the art. See,e.g., Supplementary Table S2 and Supplementary Figure S2 of Fonfara etal., Phylogeny of Cas9 determines functional exchangeability of dual-RNAand Cas9 among orthologous type II CRISPR-Cas systems, Nucl. Acids Res.2013, doi: 10.1093/nar/gkt1074, which are incorporated herein byreference in their entirety. Cas9 variants of the sequences providedherein or known in the art comprising one or more mutations, e.g., atleast one, at least two, at least three, at least four, at least five,at least six, or at least seven mutations as provided herein, e.g., ofone or more amino acid residue that is homologous to amino acid residues50, 86, 115, 108, 141, 175, 217, 230, 257, 261, 262, 267, 274, 284, 294,331, 319, 324, 341, 388, 405, 409, 435, 461, 466, 480, 510, 522, 543,548, 593, 653, 673, 694, 711, 712, 715, 772, 777, 798, 811, 839, 847,955, 967, 991, 1063, 1139, 1199, 1207, 1219, 1224, 1227, 1229, 1256,1264, 1296, 1318, 1356, and/or 1362 in SEQ ID NO: 9 are provided by thisdisclosure, for example, Cas9 variants comprising a A262T, K294R, S409I,E480K, E543D, M694I, and/or E1219V mutation.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of theembodiments described herein. The scope of the present disclosure is notintended to be limited to the above description, but rather is as setforth in the appended claims.

Articles such as “a,” “an,” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between two or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present, unless indicated to the contrary or otherwiseevident from the context. The disclosure of a group that includes “or”between two or more group members provides embodiments in which exactlyone member of the group is present, embodiments in which more than onemembers of the group are present, and embodiments in which all of thegroup members are present. For purposes of brevity those embodimentshave not been individually spelled out herein, but it will be understoodthat each of these embodiments is provided herein and may bespecifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations,combinations, and permutations in which one or more limitation, element,clause, or descriptive term, from one or more of the claims or from oneor more relevant portion of the description, is introduced into anotherclaim. For example, a claim that is dependent on another claim can bemodified to include one or more of the limitations found in any otherclaim that is dependent on the same base claim. Furthermore, where theclaims recite a composition, it is to be understood that methods ofmaking or using the composition according to any of the methods ofmaking or using disclosed herein or according to methods known in theart, if any, are included, unless otherwise indicated or unless it wouldbe evident to one of ordinary skill in the art that a contradiction orinconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that every possible subgroup of the elements is alsodisclosed, and that any element or subgroup of elements can be removedfrom the group. It is also noted that the term “comprising” is intendedto be open and permits the inclusion of additional elements or steps. Itshould be understood that, in general, where an embodiment, product, ormethod is referred to as comprising particular elements, features, orsteps, embodiments, products, or methods that consist, or consistessentially of, such elements, features, or steps, are provided as well.For purposes of brevity those embodiments have not been individuallyspelled out herein, but it will be understood that each of theseembodiments is provided herein and may be specifically claimed ordisclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in some embodiments, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.For purposes of brevity, the values in each range have not beenindividually spelled out herein, but it will be understood that each ofthese values is provided herein and may be specifically claimed ordisclaimed. It is also to be understood that unless otherwise indicatedor otherwise evident from the context and/or the understanding of one ofordinary skill in the art, values expressed as ranges can assume anysubrange within the given range, wherein the endpoints of the subrangeare expressed to the same degree of accuracy as the tenth of the unit ofthe lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

1-51. (canceled)
 52. A polynucleotide encoding a fusion proteincomprising: (i) a nucleic acid programmable DNA binding protein(napDNAbp) domain; (ii) a cytidine deaminase domain; and (iii) a Gamprotein.
 53. The polynucleotide of claim 52, wherein the fusion proteinfurther comprises (iv) a first uracil glycosylase inhibitor (UGI)domain.
 54. The polynucleotide of claim 52, wherein the nucleic acidprogrammable DNA binding protein (napDNAbp) domain is a Cas9 domain. 55.The polynucleotide of claim 54, wherein the Cas9 domain is a nucleaseactive Cas9 domain, a Cas9 nickase (nCas9) domain, or a nucleaseinactive Cas9 (dCas9) domain.
 56. The polynucleotide of claim 54,wherein the Cas9 domain is an nCas9 domain that comprises a D10Amutation relative to the amino acid sequence of SEQ ID NO:
 6. 57. Thepolynucleotide of claim 54, wherein the Cas9 domain comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 311 or
 317. 58. The polynucleotide of claim 53, whereinthe fusion protein further comprises a second UGI domain.
 59. Thepolynucleotide of claim 58, wherein at least one of the first UGI domainand the second UGI domain comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO:
 362. 60. The polynucleotide of claim52, wherein the cytidine deaminase domain is a deaminase from theapolipoprotein B mRNA-editing complex (APOBEC) family deaminases. 61.The polynucleotide of claim 52, wherein the cytidine deaminase domaincomprises an amino acid sequence that is at least 90% identical to anyone of the amino acid sequences of SEQ ID NOs: 323-361.
 62. Thepolynucleotide of claim 52, wherein the cytidine deaminase domaincomprises an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO:
 349. 63. The polynucleotide of claim53, wherein the fusion protein comprises the structure: NH₂-[Gamprotein]-[cytidine deaminase domain]-[napDNAbp domain]-[first UGIdomain]-COOH, and wherein each instance of “]-[” comprises an optionallinker.
 64. The polynucleotide of claim 57, wherein the fusion proteincomprises the structure: NH₂-[Gam protein]-[cytidine deaminasedomain]-[napDNAbp domain]-[first UGI domain]-[second UGI domain]-COOH,and wherein each instance of “]-[” comprises an optional linker.
 65. Thepolynucleotide of claim 52, wherein the Gam protein comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO:
 9. 66. The polynucleotide of claim 52, wherein the Gamprotein domain comprises the amino acid sequence of SEQ ID NO:
 9. 67.The polynucleotide of claim 52, wherein the fusion protein comprises theamino acid that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 394 or
 396. 68. The polynucleotide of claim 52, wherein thefusion protein comprises the amino acid sequence of SEQ ID NO: 394 or396.
 69. A vector comprising the polynucleotide of claim
 52. 70. Thevector of claim 69, wherein the vector comprises a heterologous promoterdriving expression of the polynucleotide.
 71. A cell comprising thepolynucleotide of claim
 52. 72. A pharmaceutical composition comprisingthe polynucleotide of claim 52 and a pharmaceutically acceptableexcipient.
 73. A method comprising: contacting a cell containing amutation to be corrected with the vector of claim 69 and a single guideRNA (sgRNA) configured to target the fusion protein to the mutation. 74.The method of claim 73, wherein the cell is obtained from a subjecthaving a disease associated with or caused by the mutation.
 75. Themethod of claim 73, wherein the mutation is a point mutation.